U.S. patent number 9,670,286 [Application Number 15/169,903] was granted by the patent office on 2017-06-06 for disease therapy by inducing immune response to trop-2 expressing cells.
This patent grant is currently assigned to IBC Pharmaceuticals, Inc.. The grantee listed for this patent is IBC Pharmaceuticals, Inc.. Invention is credited to Chien-Hsing Chang, David M. Goldenberg, Diane Rossi, Edmund A. Rossi.
United States Patent |
9,670,286 |
Chang , et al. |
June 6, 2017 |
Disease therapy by inducing immune response to Trop-2 expressing
cells
Abstract
The present invention concerns compositions and methods of use
of bispecific antibodies comprising at least one binding site for
Trop-2 (EGP-1) and at least one binding site for CD3. The
bispecific antibodies are of use for inducing an immune response
against a Trop-2 expressing tumor, such as carcinoma of the
esophagus, pancreas, lung, stomach, colon, rectum, urinary bladder,
breast, ovary, uterus, kidney or prostate. The methods may
comprising administering the bispecific antibody alone, or with one
or more therapeutic agents such as antibody-drug conjugates,
interferons (preferably interferon-.alpha.), and/or checkpoint
inhibitor antibodies. The bispecific antibody is capable of
targeting effector T cells, NK cells, monocytes or neutrophils to
induce leukocyte-mediated cytotoxicity of Trop-2.sup.+ cancer
cells. The cytotoxic immune response is enhanced by
co-administration of interferon, checkpoint inhibitor antibody
and/or ADC.
Inventors: |
Chang; Chien-Hsing
(Downingtown, PA), Goldenberg; David M. (Mendham, NJ),
Rossi; Edmund A. (Woodland Park, NJ), Rossi; Diane
(Woodland Park, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
IBC Pharmaceuticals, Inc. |
Morris Plains |
NJ |
US |
|
|
Assignee: |
IBC Pharmaceuticals, Inc.
(Morris Plains, NJ)
|
Family
ID: |
53043966 |
Appl.
No.: |
15/169,903 |
Filed: |
June 1, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20160264678 A1 |
Sep 15, 2016 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14600560 |
Jan 20, 2015 |
9382329 |
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14106737 |
Dec 14, 2013 |
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13966450 |
Apr 19, 2016 |
9315567 |
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61942752 |
Feb 21, 2014 |
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62049826 |
Sep 12, 2014 |
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61807998 |
Apr 3, 2013 |
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61733268 |
Dec 4, 2012 |
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61682965 |
Aug 14, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
47/6863 (20170801); A61K 39/39558 (20130101); C07K
16/2887 (20130101); A61K 47/6857 (20170801); A61K
47/6851 (20170801); C07K 16/3007 (20130101); A61K
38/21 (20130101); A61K 38/212 (20130101); A61K
47/6803 (20170801); A61K 47/6859 (20170801); C07K
16/2803 (20130101); C07K 16/2833 (20130101); A61K
39/3955 (20130101); C07K 16/2863 (20130101); C07K
16/2809 (20130101); A61K 45/06 (20130101); C07K
16/44 (20130101); C07K 16/30 (20130101); A61K
31/4745 (20130101); A61K 47/60 (20170801); A61K
39/39558 (20130101); A61K 2300/00 (20130101); A61K
38/21 (20130101); A61K 2300/00 (20130101); A61K
2039/505 (20130101); C07K 2317/31 (20130101); C07K
2317/732 (20130101); C07K 2317/51 (20130101); C07K
2317/54 (20130101); C07K 2317/24 (20130101); C07K
2317/565 (20130101); C07K 2317/515 (20130101); C07K
2317/77 (20130101); C07K 2317/622 (20130101); C07K
2317/73 (20130101); C07K 2317/55 (20130101); A61K
2039/507 (20130101) |
Current International
Class: |
C07K
16/28 (20060101); C07K 16/30 (20060101); A61K
38/21 (20060101); C07K 16/44 (20060101); A61K
45/06 (20060101); A61K 39/395 (20060101); A61K
31/4745 (20060101); A61K 39/00 (20060101) |
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|
Primary Examiner: Canella; Karen
Attorney, Agent or Firm: Nakashima; Richard A.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 14/600,560 (now issued U.S. Pat. No. 9,382,329), which claimed
the benefit under 35 U.S.C. 119(e) of provisional U.S. Patent
Application No. 61/942,752, filed Feb. 21, 2014, and 62/049,826,
filed Sep. 12, 2104. U.S. Ser. No. 14/600,560 was a
continuation-in-part of U.S. patent application Ser. No.
14/106,737, filed Dec. 14, 2013, which was a continuation-in-part
of U.S. patent application Ser. No. 13/966,450 (now U.S. issued
U.S. Pat. No. 9,315,567), filed Aug. 14, 2013, which claimed the
benefit under 35 U.S.C. 119(e) of provisional U.S. Patent
Applications 61/682,965, filed Aug. 14, 2012; 61/733,268, filed
Dec. 4, 2012, and 61/807,998, filed Apr. 3, 2013. Each priority
application is incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A method of inducing an immune response to a Trop-2 expressing
cancer comprising: a) administering to a subject with a Trop-2
expressing cancer a bispecific antibody that comprises (i) at least
one anti-Trop-2 antibody or antigen-binding fragment thereof and
one anti-CD3 antibody or antigen-binding fragment thereof, wherein
the at least one anti-Trop-2 antibody or fragment thereof binds to
the same epitope as an anti-Trop-2 antibody comprising the light
chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:115); CDR2
(SASYRYT, SEQ ID NO:116); and CDR3 (QQHYITPLT, SEQ ID NO:117) and
the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:118); CDR2
(WINTYTGEPTYTDDFKG, SEQ ID NO:119) and CDR3 (GGFGSSYWYFDV, SEQ ID
NO:120); and b) administering to the subject at least one
therapeutic agent selected from the group consisting of (i)
interferon .alpha.; and (ii) a checkpoint inhibitor antibody.
2. The method of claim 1, wherein the checkpoint inhibitor antibody
is selected from the group consisting of lambrolizumab (MK-3475),
nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105,
MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirilumab, IPH2101 and
tremelimumab.
3. The method of claim 1, wherein the checkpoint inhibitor antibody
binds to an antigen selected from the group consisting of CTLA4,
PD1, PD-L1, LAG3, B7-H3, B7-H4, KIR and TIM3.
4. The method of claim 1, wherein the bispecific antibody and the
therapeutic agent are administered simultaneously.
5. The method of claim 1, wherein the interferon-.alpha. is
administered as free interferon, PEGylated interferon, an
interferon fusion protein or interferon conjugated to an
antibody.
6. The method of claim 1, wherein the bispecific antibody comprises
at least one antibody fragment selected from the group consisting
of a scFv, a Fab and a dAb.
7. The method of claim 1, wherein the Trop-2 expressing cancer is a
carcinoma of the esophagus, pancreas, lung, stomach, colon, rectum,
urinary bladder, breast, ovary, uterus, kidney or prostate.
8. The method of claim 1, further comprising administering to the
subject an anti-cancer agent selected from the group consisting of
a second antibody or antigen-binding fragment thereof, a drug, a
toxin, an enzyme, an anti-angiogenic agent, a pro-apoptotic agent,
an antibiotic, a hormone, an immunomodulator, a cytokine, a boron
compound and a radioisotope.
9. The method of claim 1, wherein the bispecific antibody comprises
a humanized RS7 (anti-Trop-2) antibody or antigen-binding fragment
thereof.
10. The method of claim 1, wherein the bispecific antibody
comprises an Okt3 (anti-CD3) antibody or antigen-binding fragment
thereof.
11. The method of claim 1, wherein the bispecific antibody
comprises the amino acid sequence of SEQ ID NO:107.
12. The method of claim 1, wherein the bispecific antibody induces
an immune response to a Trop-2 expressing cancer without increasing
cytokine production to levels capable of inducing cytokine release
syndrome (CRS).
13. The method of claim 1, wherein the bispecific antibody induces
trogocytosis of cell surface antigens between Trop-2 expressing
cancer cells and T cells.
14. The method of claim 1, wherein the bispecific antibody and the
therapeutic agent are administered sequentially.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Dec. 29,
2014, is named IBC140US1_SL and is 62,950 bytes in size.
FIELD
The present invention concerns compositions and methods of use of
bispecific antibodies targeting Trop-2 and CD3, that are capable of
inducing an immune response against Trop-2 expressing cells, such
as Trop-2.sup.+ cancer cells. Preferably, the bispecific antibody
is administered in combination with one or more other therapeutic
agents, such as an antibody-drug conjugate, an interferon such as
such as interferon-.alpha., interferon-.beta. or
interferon-.lamda., or a checkpoint inhibitor antibody. More
preferably, the bispecific antibody is an
anti-Trop-2.times.anti-CD3 antibody that is administered in
combination with interferon-.alpha.. Most preferably, the
anti-Trop-2 antibody is a hRS7 antibody. The compositions and
methods are of use to treat Trop-2.sup.+ tumors, such as carcinomas
of the esophagus, pancreas, lung, stomach, colon and rectum,
urinary bladder, breast, ovary, uterus, kidney and prostate, more
preferably pancreatic cancer or gastric cancer. In preferred
embodiments, the bispecific antibody is made as a DOCK-AND-LOCK.TM.
complex, in which the components are attached together using the
binding interaction between dimerization and docking domain (DDD)
moieties from human protein kinase A (PKA) regulatory subunits and
anchor domain (AD) moieties from AKAPs (A-kinase anchor proteins).
However, other methods of making bispecific antibody complexes are
known and may be used. The bispecific antibody redirects effector T
cells, monocytes, NK cells or neutrophils to target diseased cells
or tissues and induces an immune response against the target.
BACKGROUND
Use of bispecific antibodies (bsAbs) to redirect effector T cells
for the targeted killing of tumor cells has shown considerable
promise both pre-clinically and clinically (see, e.g., Topp et al.,
2012, Blood 120:5185-87; Bargou et al., 2008, Science 321:974-77).
The bispecific antibodies developed to date contain a first binding
site specific to CD3 for T-cell recruitment and activation and a
second binding site for a targeted disease-associated antigen, such
as CD19 (Bassan, 2012, Blood 120:5094-95). The bispecific antibody
brings CD3.sup.+ T cells into direct contact with targeted disease
cells and induces cell-mediated cytotoxicity (Bassan, 2012).
Anti-CD3.times.anti-CD19 bispecific antibodies have been reported
to produce a complete and durable molecular remission at very low
concentrations in approximately 70% of adult patients with
MRD.sup.+ ALL (Topp et al., 2012, Blood 120:5185-87). Bispecific
antibodies recognizing gliomas and the CD3 epitope on T cells have
been successfully used in treating brain tumors in human patients
(Nitta, et al. Lancet 1990; 355:368-371).
Leukocyte redirecting bsAbs are not limited to T cells. The
bispecific killer engagers (BiKEs) comprising scFvs against the NK
cell antigen CD16 and a tumor-associated antigen (e.g., CD19, CD22,
CD33) have also shown potent anti-cancer activity (e.g., Miller,
Hematology Soc Hematol Educ Pogram 2013:247-53). Other alternatives
include trispecific killer engagers (TriKEs), such as
anti-CD16.times.anti-CD19.times.anti-CD22 (Miller, 2013; Gleason et
al., 2012, Mol Cancer Ther 11:2674-84). An
anti-CD16.times.anti-CD33 BiKE was used to treat AML and
myelodysplastic syndrome (Miller, 2013; Wiernik et al., 2013, Clin
Cancer Res 19:3844-55). In refractory AML, a CD16.times.CD33 BiKE
led to potent tumor cell killing and cytokine production by NK
cells. Inhibition of ADAM17 enhanced the CD16.times.CD33 BiKE
response (Miller, 2013). Other trispecific, trivalent constructs,
for example against CD16/CD19/HLA-DR, have been reported (Schubert
et al., 2012, mAbs 4:45-56).
Numerous methods to produce bispecific antibodies are known (see,
e.g. U.S. Pat. No. 7,405,320). Bispecific antibodies can be
produced by the quadroma method, which involves the fusion of two
different hybridomas, each producing a monoclonal antibody
recognizing a different antigenic site (Milstein and Cuello, Nature
1983; 305:537-540). The fused hybridomas are capable of
synthesizing two different heavy chains and two different light
chains, which can associate randomly to give a heterogeneous
population of 10 different antibody structures of which only one of
them, amounting to 1/8 of the total antibody molecules, will be
bispecific, and therefore must be further purified from the other
forms. Fused hybridomas are often less stable cytogenetically than
the parent hybridomas, making the generation of a production cell
line more problematic.
Another method for producing bispecific antibodies uses
heterobifunctional cross-linkers to chemically tether two different
monoclonal antibodies, so that the resulting hybrid conjugate will
bind to two different targets (Staerz, et al. Nature 1985;
314:628-631; Perez, et al. Nature 1985; 316:354-356). Bispecific
antibodies generated by this approach are essentially
heteroconjugates of two IgG molecules, which diffuse slowly into
tissues and are rapidly removed from the circulation. Bispecific
antibodies can also be produced by reduction of each of two
parental monoclonal antibodies to the respective half molecules,
which are then mixed and allowed to reoxidize to obtain the hybrid
structure (Staerz and Bevan. Proc Natl Acad Sci USA 1986;
83:1453-1457). An alternative approach involves chemically
cross-linking two or three separately purified Fab' fragments using
appropriate linkers. All these chemical methods are undesirable for
commercial development due to high manufacturing cost, laborious
production process, extensive purification steps, low yields
(<20%), and heterogeneous products.
Discrete V.sub.H and V.sub.L domains of antibodies produced by
recombinant DNA technology may pair with each other to form a dimer
(recombinant Fv fragment) with binding capability (U.S. Pat. No.
4,642,334). However, such non-covalently associated molecules are
not sufficiently stable under physiological conditions to have any
practical use. Cognate V.sub.H and V.sub.L domains can be joined
with a peptide linker of appropriate composition and length
(usually consisting of more than 12 amino acid residues) to form a
single-chain Fv (scFv) with binding activity. Methods of
manufacturing scFv-based agents of multivalency and
multispecificity by varying the linker length were disclosed in
U.S. Pat. No. 5,844,094, U.S. Pat. No. 5,837,242 and WO 98/44001.
Common problems that have been frequently associated with
generating scFv-based agents of multivalency and multispecificity
are low expression levels, heterogeneous products, instability in
solution leading to aggregates, instability in serum, and impaired
affinity.
Several bispecific antibodies targeting CD3 and CD19 are in
clinical development. An scFv-based bispecific antibody construct,
known as BITE.RTM. (Bispecific T-cell Engager), employs a single
polypeptide containing 2 antigen-binding specificities, each
contributed by a cognate VH and VL, linked in tandem via a flexible
linker (see, e.g., Nagorsen et al., 2009, Leukemia & Lymphoma
50:886-91; Amann et al., 2009, J Immunother 32:453-64; Baeuerle and
Reinhardt, 2009, Cancer Res 69:4941-44). Another bispecific
antibody called DART.RTM. (Dual-Affinity Re-Targeting) utilizes a
disulfide-stabilized diabody design (see, e.g., Moore et al., 2011,
Blood 117:4542-51; Veri et al., 2010, Arthritis Rheum 62:1933-43).
Both BITE.RTM. and DART.RTM. exhibit fast blood clearance due to
their small size (.about.55 kDa), which requires frequent
administration to maintain therapeutic levels of the bispecific
antibodies.
Interferons are critical role players in the antitumor and
antimicrobial host defense, and have been extensively explored as
therapeutic agents for cancer and infectious disease (Billiau et
al., 2006, Cytokine Growth Factor Rev 17:381-409; Pestka et al.,
2004, Immunol Rev 202:8-32). Despite considerable efforts with type
I and II interferons (IFN-.alpha./.beta. and .gamma.), their use in
clinic settings have been limited because of the short circulation
half-life, systemic toxicity, and suboptimal responses in patients
(Pestka et al., 2004, Immunol Rev 202:8-32; Miller et al., 2009,
Ann N Y Acad Sci 1182:69-79). The discovery of the IFN-.lamda.
family in early 2003 brought an exciting new opportunity to develop
alternative IFN agents for these unmet clinical indications
(Kotenko et al., 2003, Nat Immunol 4:69-77; Sheppard et al., 2003,
Nat Immunol 4:63-8).
The therapeutic effectiveness of IFNs has been validated to date by
the approval of IFN-.alpha.2 for treating hairy cell leukemia,
chronic myelogenous leukemia, malignant melanoma, follicular
lymphoma, condylomata acuminata, AIDs-related Kaposi sarcoma, and
chronic hepatitis B and C; IFN-.beta. for treating multiple
sclerosis; and IFN-.gamma. for treating chronic granulomatous
disease and malignant osteopetrosis. Despite a vast literature on
this group of autocrine and paracrine cytokines, their functions in
health and disease are still being elucidated, including more
effective and novel forms being introduced clinically (Pestka,
2007, J. Biol. Chem 282:20047-51; Vilcek, 2006, Immunity
25:343-48). The effects of combination of various interferons with
antibody-based therapies also remain under investigation.
Antibody-drug conjugates (ADCs) are a potent class of therapeutic
constructs that allow targeted delivery of cytotoxic agents to
target cells, such as cancer cells. Because of the targeting
function, these compounds show a much higher therapeutic index
compared to the same systemically delivered agents. ADCs have been
developed as intact antibodies or antibody fragments, such as
scFvs. The antibody or fragment is linked to one or more copies of
drug via a linker that is stable under physiological conditions,
but that may be cleaved once inside the target cell. ADCs approved
for therapeutic use include gemtuzumab ozogamicin for AML
(subsequently withdrawn from the market), brentuximab vedotin for
ALCL and Hodgkin lymphoma, and trastuzumab emtansine for
HER2-positive metastatic breast cancer (Verma et al., 2012, N Engl
J Med 367:1783-91; Bross et al., 2001, Clin Cancer Res 7:1490-96;
Francisco et al., 2003, Blood 102:1458-65). Numerous other
candidate ADCs are currently in clinical testing, such as
inotuzumab ozogamicin (Pfizer), glembatumomab vedotin (Celldex
Therapeutics), SAR3419 (Sanofi-Aventis), SAR56658 (Sanofi-Aventis),
AMG-172 (Amgen), AMG-595 (Amgen), BAY-94-9343 (Bayer), BIIB015
(Biogen Idec), BT062 (Biotest), SGN-75 (Seattle Genetics),
SGN-CD19A (Seattle Genetics), vorsetuzumab mafodotin (Seattle
Genetics), ABT-414 (AbbVie), ASG-5ME (Agensys), ASG-22ME (Agensys),
ASG-16M8F (Agensys), IMGN-529 (ImmunoGen), IMGN-853 (ImmunoGen),
MDX-1203 (Medarex), MLN-0264 (Millenium), RG-7450
(Roche/Genentech), RG-7458 (Roche/Genentech), RG-7593
(Roche/Genentech), RG-7596 (Roche/Genentech), RG-7598
(Roche/Genentech), RG-7599 (Roche/Genentech), RG-7600
(Roche/Genentech), RG-7636 (Roche/Genentech), anti-PSMA ADC
(Progenics), lorvotuzumab mertansine (ImmunoGen),
milatuzumab-doxorubicin (Immunomedics), IMMU-130 (Immunomedics),
IMMU-132 (Immunomedics) and antibody conjugates of
pro-2-pyrrolinodoxorubicin. (See, e.g., Li et al., 2013, Drug Disc
Ther 7:178-84; Firer & Gellerman, J Hematol Oncol 5:70; Beck et
al., 2010, Discov Med 10:329-39; Mullard, 2013, Nature Rev Drug
Discovery 12:329, Provisional U.S. Patent Application 61/761,845.)
Because of the potential of ADCs to act as potent anti-cancer
agents with reduced systemic toxicity, they may be used either
alone or as an adjunct therapy to reduce tumor burden.
Another promising approach to immunotherapy concerns use of
antagonistic antibodies against immune checkpoint proteins (e.g.,
Pardoll, 2012, Nature Reviews Cancer 12:252-64). Immune checkpoints
function as endogenous inhibitory pathways for immune system
function that act to maintain self-tolerance and to modulate the
duration and extent of immune response to antigenic stimulation
(Pardoll, 2012). However, it appears that tumor tissues and
possibly certain pathogens may co-opt the checkpoint system to
reduce the effectiveness of host immune response, resulting in
tumor growth and/or chronic infection (see, e.g., Pardoll, 2012,
Nature Reviews Cancer 12:252-64; Nirschl & Drake, 2013, Clin
Cancer Res 19:4917-24). Checkpoint molecules include CTLA4
(cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death
protein 1), PD-L1 (programmed cell death ligand 1), LAG-3
(lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and
mucin protein-3) and several others (Pardoll, 2012, Nature Reviews
Cancer 12:252-64; Nirschl & Drake, 2013, Clin Cancer Res
19:4917-24). Antibodies against several of the checkpoint proteins
(CTLA4, PD1, PD-L1) are in clinical trials and has shown unexpected
efficacy against tumors that were resistant to standard
treatments.
A need exists for methods and compositions to generate improved
bispecific antibody complexes with longer T.sub.1/2, better
pharmacokinetic properties, increased in vivo stability and/or
improved in vivo efficacy. A further need exists for combination
therapies to improve efficacy of treatments directed to inducing
immune response against various diseases, such as Trop-2.sup.+
cancer.
SUMMARY
The present invention relates to bispecific antibodies of use to
treat diseases involving Trop-2+ cells, such as Trop-2.sup.+ cancer
cells. Trop-2 is overexpressed in numerous types of solid tumors,
such as carcinomas of the esophagus, pancreas, lung, stomach, colon
and rectum, urinary bladder, breast, ovary, uterus, cervix, kidney
and prostate. Preferably, the bispecific antibody is of use to
treat gastric cancer or pancreatic cancer. Administration of the
bispecific antibody induces an immune response to cells that are
Trop-2.sup.+. Although Trop-2 is also expressed in some normal
tissues (e.g., Stepan et al., 2011, J Histochem Cytochem
59:701-10), the Examples below demonstrate that anti-Trop-2
antibodies may be administered in vivo in both animal model systems
and human subjects, with only tolerable toxicities. In other
preferred embodiments, administration of bispecific antibody to a
subject induces an immune response against Trop-2.sup.+ cancer
cells without elevating levels of cytokines that would induce
cytokine release syndrome (CRS). In alternative preferred
embodiments, the bispecific antibody induces trogocytosis of cell
surface antigens between Trop-2.sup.+ cancer cells and T cells.
In preferred embodiments, the bispecific antibody contains binding
sites for Trop-2 and for CD3. However, other T cell or leukocyte
antigens may be targeted besides CD3. Exemplary T-cell antigens are
selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD8,
CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90. Exemplary
antigens expressed on NK cells are selected from the group
consisting of CD8, CD16, CD56, CD57, ADAM17, KIR and CD137.
Exemplary monocyte antigens are selected from the group consisting
of CD74, HLA-DR alpha chain, CD14, CD16, CD64 and CD89. Exemplary
neutrophil antigens are selected from the group consisting of
CEACAM6, CEACAM8, CD16b, CD32a, CD89, CD177, CD11a, CD11b and
SLC44A2. Preferably the T-cell antigen is CD3, or the NK cell
antigen is CD16.
In alternative embodiments, other tumor-associated antigens besides
Trop-2 may be targeted. Tumor-associated antigens that may be
targeted include, but are not limited to, alpha-fetoprotein (AFP),
.alpha.-actinin-4, A3, antigen specific for A33 antibody, ART-4,
B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic
anhydrase IX, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4,
CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22,
CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44,
CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70,
CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD132, CD133,
CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA4, CXCR4, CXCR7,
CXCL12, HIF-1.alpha., colon-specific antigen-p (CSAp), CEA
(CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2),
EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1,
Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-.beta.,
HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its
subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1),
HSP70-2M, HST-2, Ia, IGF-1R, IFN-.gamma., IFN-.alpha., IFN-.beta.,
IFN-.lamda., IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2,
IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like
growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y,
LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE,
MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A,
MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2,
MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreatic cancer mucin,
PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid
phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25,
RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72,
tenascin, TRAIL receptors, TNF-.alpha., Tn antigen,
Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B
fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b,
C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene
marker and an oncogene product (see, e.g., Sensi et al., Clin
Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol 2007,
178:1975-79; Novellino et al. Cancer Immunol Immunother 2005,
54:187-207).
Exemplary anti-TAA antibodies that may be used include, but are not
limited to, hA19 (anti-CD19, U.S. Pat. No. 7,109,304), hR1
(anti-IGF-1R, U.S. patent application Ser. No. 12/722,645, filed
Mar. 12, 2010), hPAM4 (anti-MUC5ac, U.S. Pat. No. 7,282,567), hA20
(anti-CD20, U.S. Pat. No. 7,251,164), hIMMU31 (anti-AFP, U.S. Pat.
No. 7,300,655), hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2
(anti-CD22, U.S. Pat. No. 7,074,403), hMu-9 (anti-CSAp, U.S. Pat.
No. 7,387,773), hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180),
hMN-14 (anti-CEACAM5, U.S. Pat. No. 6,676,924), hMN-15
(anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7 (anti-EGP-1, U.S.
Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat. No. 7,541,440),
Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496), the Examples
section of each cited patent or application incorporated herein by
reference.
Alternative antibodies that may be used for treatment of various
disease states include, but are not limited to, abciximab
(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab
(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33),
ibritumomab (anti-CD20), panitumumab (anti-EGFR), rituximab
(anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-ErbB2),
lambrolizumab (anti-PD1 receptor), nivolumab (anti-PD1 receptor),
ipilimumab (anti-CTLA4), abagovomab (anti-CA-125), adecatumumab
(anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab
(anti-CD125), obinutuzumab (GA101, anti-CD20), CC49 (anti-TAG-72),
AB-PG1-XG1-026 (anti-PSMA, U.S. patent application Ser. No.
11/983,372, deposited as ATCC PTA-4405 and PTA-4406), D2/B
(anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6 receptor),
basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab
(anti-CD11a), GA101 (anti-CD20; Glycart Roche), atalizumab
(anti-.alpha.4 integrin), omalizumab (anti-IgE); anti-TNF-.alpha.
antibodies such as CDP571 (Ofei et al., 2011, Diabetes 45:881-85),
MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (Thermo Scientific,
Rockford, Ill.), infliximab (Centocor, Malvern, Pa.), certolizumab
pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,
Belgium), adalimumab (Abbott, Abbott Park, Ill.), BENLYSTA.RTM.
(Human Genome Sciences); anti-CD38 antibodies such as MOR03087
(MorphoSys AG), MOR202 (Celgene), HuMax-CD38 (Genmab) or
daratumumab (Johnson & Johnson).
Preferably, the bispecific antibody is administered in combination
with one or more other therapeutic agents, such as antibodies,
antibody fragments, peptides, drugs, toxins, chemotherapeutic
agents, enzymes, nucleases, hormones, immunomodulators, antisense
oligonucleotides, small interfering RNA (siRNA), chelators, boron
compounds, photoactive agents, dyes, and radioisotopes. More
preferably, the additional therapeutic agent is an antibody-drug
conjugate, an interferon such as such as interferon-.alpha.,
interferon-.beta. or interferon-.lamda., or an antagonistic
checkpoint inhibitor antibody. Most preferably, the therapeutic
agent is interferon-.alpha..
An exemplary design for a leukocyte redirecting bsAb disclosed in
the Examples below combined an anti-CD3 scFv with an anti-CD19
F(ab).sub.2 to form a construct designated (19)-3s, which
specifically targeted B cells. Other bsAbs combining anti-CD3 with
antibody fragments against other tumor-associated antigens,
discussed in more detail below, are of use in targeted leukocyte
immunotherapy of various solid tumors. The advantages of this
design include bivalent binding to tumor cells, a larger size
(.about.130 kDa) to preclude rapid renal clearance, and potent
leukocyte mediated cytotoxicity. The bsAbs mediate the formation of
immunological synapses between leukocytes and cognate target cells,
induce leukocyte activation and proliferation in the presence of
target cells, redirect potent leukocyte mediated killing of target
cells in vitro and inhibit growth of human tumors in vivo.
A preferred embodiment concerns leukocyte redirecting bispecific
antibodies produced as trivalent DNL.TM. complexes, with longer
T.sub.1/2, better pharmacokinetic properties and increased in vivo
stability. Methods for production and use of DNL.TM. complexes,
comprising dimers of DDD moieties from human PKA regulatory
subunits RI.alpha., RI.beta., RII.alpha. or RII.beta., bound to AD
moieties from AKAPs, are well known (see, e.g., U.S. Pat. Nos.
7,550,143; 7,521,056; 7,534,866; 7,527,787; 7,666,400; 7,906,118;
7,901,680; 8,003,111 and 8,034,352, the Examples section of each
incorporated herein by reference.) By attaching different effector
moieties, such as antibodies or antibody fragments, to the DDD and
AD moieties, DNL.TM. complexes comprising virtually any combination
of effectors may be constructed and used.
The antibodies of use can be of various isotypes, preferably human
IgG1, IgG2, IgG3 or IgG4, more preferably comprising human IgG1
hinge and constant region sequences. The antibodies or fragments
thereof can be chimeric human-mouse, humanized (human framework and
murine hypervariable (CDR) regions), or fully human, as well as
variations thereof, such as half-IgG4 antibodies (referred to as
"unibodies"), as described by van der Neut Kolfschoten et al.
(Science 2007; 317:1554-1557). More preferably, the antibodies or
fragments thereof may be designed or selected to comprise human
constant region sequences that belong to specific allotypes, which
may result in reduced immunogenicity when administered to a human
subject. Preferred allotypes for administration include a non-G1m1
allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More
preferably, the allotype is selected from the group consisting of
the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.
Other preferred embodiments concern compositions and/or use of
leukocyte-redirecting complexes in combination with one or more
checkpoint inhibitor antibodies. Such antibodies are antagonistic
for checkpoint inhibitor function. Many such antibodies are known
in the art, such as lambrolizumab (MK-3475, Merck), nivolumab
(BMS-936558, Bristol-Myers Squibb), pidilizumab (CT-011, CureTech
Ltd.), AMP-224 (Merck), MDX-1105 (Medarex), MEDI4736 (MedImmune),
MPDL3280A (Genentech), BMS-936559 (Bristol-Myers Squibb),
ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer).
Anti-KIR antibodies such as lirilumab (Innate Pharma) and IPH2101
(Innate Pharma) may perform similar functions in NK cells. Any
known checkpoint inhibitor antibody may be used in combination with
one or more of the other agents. Combination therapy with
immunostimulatory antibodies has been reported to enhance efficacy,
for example against tumor cells. Morales-Kastresana et al. (2013,
Clin Cancer Res 19:6151-62) showed that the combination of
anti-PD-L1 (10B5) antibody with anti-CD137 (1D8) and anti-OX40
(OX86) antibodies provided enhanced efficacy in a transgenic mouse
model of hepatocellular carcinoma. Combination of anti-CTLA4 and
anti-PD1 antibodies has also been reported to be highly efficacious
(Wolchok et al., 2013, N Engl J Med 369:122-33). Combination of
rituximab with anti-KIR antibody, such as lirilumab (Innate Pharma)
or IPH2101 (Innate Pharma), was also more efficacious against
hematopoietic tumors (Kohrt et al., 2012). The person of ordinary
skill will realize that the subject combination therapy may include
combinations with multiple antibodies that are immunostimulatory,
anti-tumor or anti-infectious agent.
Another agent that may be used in combination is an interferon.
Interferons of use are known in the art and may include
interferon-.alpha., interferon-.beta., interferon-.lamda.1,
interferon-.lamda.2 or interferon-.lamda.3. Preferably, the
interferon is interferon-.alpha.. The subject interferon may be
administered as free interferon, PEGylated interferon, an
interferon fusion protein or interferon conjugated to an
antibody.
In alternative embodiments, one or more of the immunomodulatory
agents discussed above may be used in combination with an
antibody-drug conjugate (ADC). ADCs are particularly effective for
reducing tumor burden without significant systemic toxicity and may
act to improve the effectiveness of the immune response induced by
leukocyte retargeting bsAb, interferon and/or checkpoint inhibitor
antibody. Exemplary ADCs of use may include ADCs approved for
therapeutic use include gemtuzumab ozogamicin for AML (subsequently
withdrawn from the market), brentuximab vedotin for ALCL and
Hodgkin lymphoma, and trastuzumab emtansine for HER2-positive
metastatic breast cancer (Verma et al., 2012, N Engl J Med
367:1783-91; Bross et al., 2001, Clin Cancer Res 7:1490-96;
Francisco et al., 2003, Blood 102:1458-65). Numerous other
candidate ADCs are currently in clinical testing, such as
inotuzumab ozogamicin (Pfizer), glembatumomab vedotin (Celldex
Therapeutics), SAR3419 (Sanofi-Aventis), SAR56658 (Sanofi-Aventis),
AMG-172 (Amgen), AMG-595 (Amgen), BAY-94-9343 (Bayer), BIIB015
(Biogen Idec), BT062 (Biotest), SGN-75 (Seattle Genetics),
SGN-CD19A (Seattle Genetics), vorsetuzumab mafodotin (Seattle
Genetics), ABT-414 (AbbVie), ASG-5ME (Agensys), ASG-22ME (Agensys),
ASG-16M8F (Agensys), IMGN-529 (ImmunoGen), IMGN-853 (ImmunoGen),
MDX-1203 (Medarex), MLN-0264 (Millenium), RG-7450
(Roche/Genentech), RG-7458 (Roche/Genentech), RG-7593
(Roche/Genentech), RG-7596 (Roche/Genentech), RG-7598
(Roche/Genentech), RG-7599 (Roche/Genentech), RG-7600
(Roche/Genentech), RG-7636 (Roche/Genentech), anti-PSMA ADC
(Progenics), lorvotuzumab mertansine (ImmunoGen),
milatuzumab-doxorubicin (Immunomedics), IMMU-130 (Immunomedics) and
IMMU-132 (Immunomedics). (See, e.g., Li et al., 2013, Drug Disc
Ther 7:178-84; Firer & Gellerman, J Hematol Oncol 5:70; Beck et
al., 2010, Discov Med 10:329-39; Mullard, 2013, Nature Rev Drug
Discovery 12:329.) Preferably, where an ADC is used in combination
with an immunomodulator, the ADC is administered prior to the
immunomodulator.
The subject agents may be administered in combination with one or
more other immunomodulators to enhance the immune response.
Immunomodulators may include, but are not limited to, a cytokine, a
chemokine, a stem cell growth factor, a lymphotoxin, an
hematopoietic factor, a colony stimulating factor (CSF),
erythropoietin, thrombopoietin, tumor necrosis factor-.alpha.
(TNF), TNF-.beta., granulocyte-colony stimulating factor (G-CSF),
granulocyte macrophage-colony stimulating factor (GM-CSF),
interferon-.alpha., interferon-.beta., interferon-.gamma.,
interferon-.lamda., stem cell growth factor designated "S1 factor",
human growth hormone, N-methionyl human growth hormone, bovine
growth hormone, parathyroid hormone, thyroxine, insulin,
proinsulin, relaxin, prorelaxin, follicle stimulating hormone
(FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH),
hepatic growth factor, prostaglandin, fibroblast growth factor,
prolactin, placental lactogen, OB protein, mullerian-inhibiting
substance, mouse gonadotropin-associated peptide, inhibin, activin,
vascular endothelial growth factor, integrin, NGF-.beta.,
platelet-growth factor, TGF-.alpha., TGF-.beta., insulin-like
growth factor-I, insulin-like growth factor-II, macrophage-CSF
(M-CSF), IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin,
endostatin, or lymphotoxin. In certain embodiments, a
leukocyte-redirecting bispecific antibody or antibody fragment may
be attached to an immunomodulator, such as a cytokine. Cytokine
complexes are disclosed, for example, in U.S. Pat. Nos. 7,906,118
and 8,034,3522, the Examples section of each incorporated herein by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and
are included to further demonstrate certain embodiments of the
present invention. The embodiments may be better understood by
reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
FIG. 1. Schematic diagram of formation of DOCK-AND-LOCK.TM. complex
comprising anti-CD19 F(ab).sub.2.times.anti-CD3 scFv.
FIG. 2A. Immune synapse formation between Daudi Burkitt lymphoma
and T cells, mediated by (19)-3s. Freshly isolated T cells were
combined with Daudi cells at an E:T ratio of 2.5:1. Cells were
treated with 0, 1 or 5 .mu.g/mL of (19)-3s for 30 min at room
temperature prior to analysis by flow cytometry. Anti-CD20-FITC and
anti-CD7-APC were used to identify Daudi and T cells, respectively.
Co-binding was indicated as the % of CD20.sup.+/CD7.sup.+ events.
After treatment with (19)-3s, 45.5% of flow events were CD20/CD7
dual-positive, indicating synapsed Daudi and T cells.
FIG. 2B. Conditions were as in FIG. 2(A), except for the absence of
(19)-3s antibody. Compared to FIG. 2(A), only 2% of flow events
were CD20/CD7 dual-positive without antibody.
FIG. 2C. Addition of (19)-3s resulted in association of >90% of
the Daudi with T cells.
FIG. 3A. Jurkat (T cells) and Daudi (B cells) were combined at a
1:1 ratio, treated with 0.1 .mu.g/mL (19)-3s for 30 minutes and
stained with anti-CD20-FITC, prior to analysis by fluorescence
microscopy.
FIG. 3B. Jurkat (T cells) and Daudi (B cells) were combined at a
1:1 ratio, treated with 0.1 .mu.g/mL (19)-3s for 30 minutes and
stained with anti-CD20-FITC and anti-CD3-PE, prior to analysis by
fluorescence microscopy.
FIG. 3C. The merged image of FIGS. 3A and 3B reveals synapse
formation between green-stained Daudi and red-stained Jurkat
cells.
FIG. 3D. Synapse formation was not evident in the absence of
(19)-3s.
FIG. 4. Dose response analysis of (19)-3s mediated cell-to-cell
association of Daudi and Jurkat cells as a function of increasing
concentrations of (19)-3s.
FIG. 5A. Comparison of cell-to-cell association mediated by
BITE.RTM. and DART.TM.. The data for BITE.RTM. and DART.TM. was
taken from Moore et al. (2011, Blood 117:4542-4551.
FIG. 5B. Comparison of cell-to-cell association mediated by
(19)-3s.
FIG. 6A. Synapse formation between T cells and Capan-1 pancreatic
cancer cells mediated by (19)-3s control bsAb. CFSE-labeled Capan-1
cells were coincubated with PKH26-labeled Jurkat in the presence of
the bsAb.
FIG. 6B. Synapse formation between T cells and Capan-1 pancreatic
cancer cells mediated by (M1)-3s MUC5AC bsAb. CFSE-labeled Capan-1
cells were coincubated with PKH26-labeled Jurkat in the presence of
the bsAb.
FIG. 6C. Synapse formation between T cells and Capan-1 pancreatic
cancer cells mediated by (E1)-3s TROP-2 targeting bsAb.
CFSE-labeled Capan-1 cells were coincubated with PKH26-labeled
Jurkat in the presence of the bsAb.
FIG. 7A. T-cell activation by (19)-3s. Upregulation of CD69
expression is an early event in T-cell activation. Daudi cells
combined with PBMCs were treated overnight with the indicated
antibodies, and stained with anti-CD3-PE and anti-CD69-APC, prior
to analysis by flow cytometry. CD69 expression was evaluated
following gating of T cells by forward vs. side scattering and
anti-CD3 staining. Combination of Daudi cells with an equal number
of PBMCs resulted in 1.6% CD69+ T cells. Addition of 3 ng/mL
(19)-3s induced 27% CD69+ T cells. Neither a control construct
[(M1)-3s], which comprises the Okt3-scFv-AD2 module fused with a
non-targeting F(ab).sub.2, nor the hA19-Fab-DDD2 module, induced
T-cell activation.
FIG. 7B. T-cell activation by (19)-3s. Daudi cells combined with
purified T cells were treated overnight with the indicated
antibodies, and stained with anti-CD3-PE and anti-CD69-APC, prior
to analysis by flow cytometry. CD69 expression was evaluated
following gating of T cells by forward vs. side scattering and
anti-CD3 staining. Treatment of Daudi and purified T cells with
(M1)-3s or hA19-Fab-DDD2 did not increase the number of CD69+ T
cells (<4%), compared to the untreated cell mixture.
Alternatively, (19)-3s induced robust T-cell activation, producing
80% CD69+ cells.
FIG. 7C. T-cell activation by (19)-3s. Purified T cells alone were
treated overnight with the indicated antibodies, and stained with
anti-CD3-PE and anti-CD69-APC, prior to analysis by flow cytometry.
CD69 expression was evaluated following gating of T cells by
forward vs. side scattering and anti-CD3 staining. Without the
addition of Daudi (target) cells, (19)-3s did not induce CD69
expression and T-cell activation. These results demonstrate that
(19)-3s-mediated synapse formation between T cells and target cells
is both required and sufficient for T-cell activation.
FIG. 8A. Induction of T-cell proliferation by (19)-3s. PBMCs were
incubated with 3 nM or 30 pM of (19)-3s, compared to IL-2/PHA
positive control and (14)-3s (non-target-binding control).
FIG. 8B. Induction of T-cell proliferation by (19)-3s. T cell
proliferation was not observed in PBMCs depleted of B cells,
indicating that target cells (B cells) are required for T-cell
activation and proliferation.
FIG. 9A. In vitro cytotoxicity of (19)-3s T-cell redirecting bsAbs.
Dose-response curves for cytotoxicity to Nalm-6, Raji, Ramos and
Namalwa cancer cells were determined for the (19)-3s DNL.TM. bsAb
complex.
FIG. 9B. In vitro cytotoxicity of (19)-3s T-cell redirecting bsAbs.
Dose-response curves for cytotoxicity to Nalm-6, Raji, Ramos and
Namalwa cancer cells were determined for the (14)-3s
(non-targeting) DNL.TM. bsAb complex.
FIG. 9C. Consistent results were observed using PBMCs, or T cells,
obtained from two different donors and Nalm-6 cancer cells.
FIG. 10A. In vitro cytotoxicity of (20)-3s, (22)-3s and (C2)-3s
T-cell redirecting bsAbs. Dose-response curves were determined for
cytotoxicity to Namalwa cells induced by (20)-3s, (22)-3s and
(C2)-3s T-cell redirecting bsAbs.
FIG. 10B. In vitro cytotoxicity of (20)-3s, (22)-3s and (C2)-3s
T-cell redirecting bsAbs. Dose-response curves were determined for
cytotoxicity to Jeko cells induced by (20)-3s, (22)-3s and (C2)-3s
T-cell redirecting bsAbs.
FIG. 10C. In vitro cytotoxicity of (20)-3s, (22)-3s and (C2)-3s
T-cell redirecting bsAbs. Dose-response curves were determined for
cytotoxicity to Daudi cells induced by (20)-3s, (22)-3s and (C2)-3s
T-cell redirecting bsAbs.
FIG. 11A. In vitro cytotoxicity of T-cell redirecting bsAbs in
solid tumor cell lines. Dose-response curves were determined for
cytotoxicity to the LS174T colon adenocarcinoma cell line for the
(14)-3s bsAb, compared to non-targeting (19)-3s bsAb.
FIG. 11B. In vitro cytotoxicity of T-cell redirecting bsAbs in
solid tumor cell lines. Dose-response curves were determined for
cytotoxicity to the Capan-1 pancreatic adenocarcinoma cell line for
the (E1)-3s bsAb, compared to non-targeting (19)-3s bsAb.
FIG. 11C. In vitro cytotoxicity of T-cell redirecting bsAbs in
solid tumor cell lines. Dose-response curves were determined for
cytotoxicity to the NCI-N87 gastric carcinoma cell line for the
(E1)-3s and (15)-3s bsAbs, compared to non-targeting (19)-3s
bsAb.
FIG. 12. Summary of in vitro cytotoxicity data for T-cell
redirecting bsAbs in cancer cell lines.
FIG. 13A. In vivo retargeting of Raji lymphoma xenografts using
(19)-3s bsAb. NOD/SCID mice bearing Raji Burkitt lymphoma
(1.times.10.sup.6 cells) xenografts, reconstituted with human PBMCs
(5.times.10.sup.6 cells) and treated with (19)-3s for only 1 week,
administered as indicated by the arrows. Control with untreated
cells.
FIG. 13B. In vivo retargeting of Raji lymphoma xenografts using
(19)-3s bsAb. NOD/SCID mice bearing Raji Burkitt lymphoma
(1.times.10.sup.6 cells) xenografts, reconstituted with human PBMCs
(5.times.10.sup.6 cells) and treated with (19)-3s for only 1 week,
administered as indicated by the arrows. Cells were treated with a
single dose of 130 .mu.g.
FIG. 13C. In vivo retargeting of Raji lymphoma xenografts using
(19)-3s bsAb. NOD/SCID mice bearing Raji Burkitt lymphoma
(1.times.10.sup.6 cells) xenografts, reconstituted with human PBMCs
(5.times.10.sup.6 cells) and treated with (19)-3s for only 1 week,
administered as indicated by the arrows. Cells were treated
3.times. with 43 .mu.g per dose.
FIG. 13D. In vivo retargeting of Raji lymphoma xenografts using
(19)-3s bsAb. NOD/SCID mice bearing Raji Burkitt lymphoma
(1.times.10.sup.6 cells) xenografts, reconstituted with human PBMCs
(5.times.10.sup.6 cells) and treated with (19)-3s for only 1 week,
administered as indicated by the arrows. Cells were treated
5.times. with 26 .mu.g per dose.
FIG. 14A. Effect of repeated dosing on in vivo retargeting of Raji
lymphoma xenografts using (19)-3s bsAb. NOD/SCID mouse xenografts
were prepared as indicated in the legend to FIG. 13. The (19)-3s
was administered as indicated by the arrows. FIG. 14A shows
untreated control.
FIG. 14B. Effect of repeated dosing on in vivo retargeting of Raji
lymphoma xenografts using (19)-3s bsAb. The (19)-3s was
administered as indicated by the arrows. Cells were treated
2.times. with 130 .mu.g per dose of (19)-3s administered i.v.
FIG. 14C. Effect of repeated dosing on in vivo retargeting of Raji
lymphoma xenografts using (19)-3s bsAb. The (19)-3s was
administered as indicated by the arrows. Cells were treated treated
2.times. with 130 .mu.g per dose of (19)-3s administered s.c.
FIG. 14D. Effect of repeated dosing on in vivo retargeting of Raji
lymphoma xenografts using (19)-3s bsAb. The (19)-3s was
administered as indicated by the arrows. Cells were treated treated
4.times. with 65 .mu.g per dose of (19)-3s administered i.v.
FIG. 14E. Effect of repeated dosing on in vivo retargeting of Raji
lymphoma xenografts using (19)-3s bsAb. The (19)-3s was
administered as indicated by the arrows. Cells were treated treated
6.times. with 43 .mu.g per dose of (19)-3s administered i.v.
FIG. 14F. Effect of repeated dosing on in vivo retargeting of Raji
lymphoma xenografts using (19)-3s bsAb. The (19)-3s was
administered as indicated by the arrows. Cells were treated treated
6.times. with 43 .mu.g per dose of control (M1)-3s administered
i.v.
FIG. 15A. In vivo efficacy of T-cell retargeting bsAbs in solid
tumor xenografts. NOD/SCID mouse xenografts were prepared with
LS174T colon adenocarcinoma. Mice were administered T cells only
without bsAb.
FIG. 15B. In vivo efficacy of T-cell retargeting bsAbs in solid
tumor xenografts. NOD/SCID mouse xenografts were prepared with
LS174T colon adenocarcinoma. Mice were treated with (E1)-3s bsAb as
indicated.
FIG. 15C. In vivo efficacy of T-cell retargeting bsAbs in solid
tumor xenografts. NOD/SCID mouse xenografts were prepared with
Capan-1 pancreatic carcinoma. Mice were administered PBMCs only
without bsAb.
FIG. 15D. In vivo efficacy of T-cell retargeting bsAbs in solid
tumor xenografts. NOD/SCID mouse xenografts were prepared with
Capan-1 pancreatic carcinoma. Mice were treated with (14)-3s bsAb
as indicated.
FIG. 16A. In vivo inhibition of tumor growth by (E1)-3s DNL.TM.
complex in the presence or absence of interferon-.alpha.. Capan-1
pancreatic carcinoma xenografts in NOD/SCID mice were treated with
anti-TROP-2.times.anti-CD3 bsAb with or without added
interferon-.alpha.. The interferon-.alpha. was added in the form of
a TROP-2 targeting DNL.TM. complex.
FIG. 16B. In vivo inhibition of tumor growth by (E1)-3s DNL.TM.
complex in the presence or absence of interferon-.alpha.. Capan-1
pancreatic carcinoma xenografts in NOD/SCID mice were treated with
anti-TROP-2.times.anti-CD3 bsAb with or without added
interferon-.alpha.. The interferon-.alpha. was added as the
commercially available PEGASYS.RTM. (peginterferon alfa-2a).
FIG. 17. Survival curves for NOD/SCID mice treated with (E1)-3s
with or without interferon-.alpha.. Controls were untreated or
treated with interferon-.alpha. alone.
FIG. 18. In vivo inhibition of tumor growth by (E1)-3s DNL.TM.
complex in the presence or absence of interferon-.alpha., compared
to TF12 control. Capan-1 pancreatic carcinoma xenografts in
NOD/SCID mice were treated with anti-TROP-2.times.anti-CD3 bsAb
with or without added interferon-.alpha., added as PEGASYS.RTM.,
compared to untreated control, TF12 control or PEGASYS.RTM.
alone.
FIG. 19. Survival curves for NOD/SCID mice treated with (E1)-3s
with or without interferon-.alpha. (PEGASYS.RTM.). Controls were
untreated or treated with PEGASYS.RTM. alone or TF12 alone.
FIG. 20. In vivo inhibition of tumor growth by (E1)-3s DNL.TM.
complex in the presence or absence of interferon-.alpha., compared
to TF12 control. NCI-N87 human gastric cancer xenografts in
NOD/SCID mice were treated with anti-TROP-2.times.anti-CD3 bsAb
with or without added interferon-.alpha., added as PEGASYS.RTM.,
compared to untreated control, TF12 control or PEGASYS.RTM.
alone.
FIG. 21. Survival curves for NOD/SCID mice with NCI-N87 gastric
cancer xenografts treated with (E1)-3s with or without
interferon-.alpha. (PEGASYS.RTM.). Controls were untreated or
treated with PEGASYS.RTM. alone or TF12 alone.
FIG. 22. Schematic representation of the nascent E1-3 polypeptide.
LP, leader peptide that is removed in mature protein; VH, heavy
chain variable domain, VK, kappa light chain variable domain, L1,
linker peptide 1; L2, linker peptide 2; L3, linker peptide 3; 6H,
hexa-histidine.
FIG. 23A. Ex vivo T cell redirected killing of BxPC3 human
pancreatic cancer solid tumor cell line.
FIG. 23B. Ex vivo T cell redirected killing of Capan-1 human
pancreatic cancer solid tumor cell line.
FIG. 23C. Ex vivo T cell redirected killing of NCI-N87 human
gastric cancer solid tumor cell line.
FIG. 24. In vivo T cell redirected therapy of NCI-N87 gastric
carcinoma in NOD-SCID mice.
FIG. 25. Immunologic synapse formation and bidirectional
trogocytosis mediated by (E1)-3s. Purified T cells were mixed with
BxPC3 cells at a 5:1 ratio and incubated for 60 minutes with 0.1
nmol/L of the indicated bsAb before staining with anti-Trop-2 MAb
C518 and GAM-Fc-FITC. The cells were analyzed by flow cytometry,
with nonconjugated T cells and BxPC3 cells first gated by forward
versus side scattering. Trogocytosis of Trop-2 from BxPC3 cells to
T cells was evident by detection of Trop-2 on T cells, specifically
in cell mixtures with (E1)-3s, shown as the percentage of
Trop-2-positive unconjugated T cells.
FIG. 26. Immunologic synapse formation and bidirectional
trogocytosis mediated by (E1)-3s. Purified T cells were mixed with
BxPC3 cells at a 5:1 ratio and incubated for 60 minutes with 0.1
nmol/L of the indicated bsAb before staining with anti-Trop-2 MAb
C518 and GAM-Fc-FITC. The cells were analyzed by flow cytometry,
with nonconjugated T cells and BxPC3 cells first gated by forward
versus side scattering. Trogocytosis resulted in a reduction of
Trop-2 on BxPC3 cells, shown as the geometric MFI.
FIG. 27A. Cytokine induction. (A) PBMCs (6.times.10.sup.6
cells/well) were combined with Raji (5.times.10.sup.5 cells/well)
and treated for 20 h with 0.1 nM 19-3 BiTE (checkered), (19)-3s
(black), or incubated without bsAb (white, not tested for D-5).
Concentrations of TNF-.alpha., IFN-.gamma., IL-2, IL-6, and IL-10
in the supernatant fluids were determined using commercial ELISA
kits. D-1 through D-8 are independent blood donors, where only D-5
was used in both A and B at the same time.
FIG. 27B. NCI-N87 cells (5.times.10.sup.5 cells/0.5 mL/well) were
cultured overnight in 24-well plates to allow cell attachment.
PBMCs were added to wells containing attached NCI-N87 cells (10:1
ratio) and treated for 20 h with 0.1 nM of (E1)-3s (black),
peginterferonalfa-2a (white), (E1)-3s plus peginterferonalfa-2a
(checkered), or untreated (gray). Concentrations of TNF-.alpha.,
IFN-.gamma., IL-2, IL-6, and IL-10 in the supernatant fluids were
determined using commercial ELISA kits. D-1 through D-8 are
independent blood donors, where only D-5 was used in both A and B
at the same time.
FIG. 28A. In-vitro cytotoxicity. Purified CD8.sup.+ T cells
isolated from a first donor were pre-treated for 24 h with 0.1 nM
peginterferonalfa-2a (.tangle-solidup., dashed), 0.1 nM 20*-2b
(.circle-solid., grey) or media (.box-solid., black) before
combining with PKH-67 green fluorescent labeled NCI-N87 cells at a
5:1 ratio. The cell mixtures were treated with titrations of
(E1)-3s for two days before counting the number of live NCI-N87
cells by flow cytometry. Non-linear regression analysis (sigmoidal
dose-response) of the percent lysis, which was calculated for each
sample using the formula: [1-(A.sub.1/A.sub.2)].times.100, where
A.sub.1 and A.sub.2 represent the number of viable target cells in
the test and untreated samples, respectively, vs the log of the
molar concentration of (E1)-3s.
FIG. 28B. In-vitro cytotoxicity. Purified CD8.sup.+ T cells
isolated from a second donor were pre-treated for 24 h with 0.1 nM
peginterferonalfa-2a (.tangle-solidup., dashed), 0.1 nM 20*-2b
(.circle-solid., grey) or media (.box-solid., black) before
combining with PKH-67 green fluorescent labeled NCI-N87 cells at a
5:1 ratio. The cell mixtures were treated with titrations of
(E1)-3s for two days before counting the number of live NCI-N87
cells by flow cytometry. Non-linear regression analysis (sigmoidal
dose-response) of the percent lysis, which was calculated for each
sample using the formula: [1-(A.sub.1/A.sub.2)].times.100, where
A.sub.1 and A.sub.2 represent the number of viable target cells in
the test and untreated samples, respectively, vs the log of the
molar concentration of (E1)-3s.
FIG. 29A. T-cell activation. Purified T cells were mixed 5:1 with
NCI-N87 cells and treated for 18 h with (E1)-3s before measuring
CD69 expression by flow cytometry. Non-linear regression analysis
(sigmoidal dose-response) of the percent CD69-positive CD4.sup.+
(.circle-solid.) or CD8.sup.+ (.box-solid.) T cells vs the log of
the molar concentration of (E1)-3s, in the presence (dashed line)
or absence (solid line) of 0.1 nM peginterferonalfa-2a.
FIG. 29B. T-cell activation. Purified T cells were mixed 5:1 with
NCI-N87 cells and treated for 18 h with (E1)-3s before measuring
CD69 expression by flow cytometry. Histogram showing anti-CD69-APC
staining of CD8.sup.+ T cells following treatment with 0.1 nM
(E1)-3s (dotted), 0.1 nM peginterferonalfa-2a (gray), or a
combination of both agents (black), in the presence of NCI-N87
cells.
FIG. 29C. T-cell activation. Purified T cells were mixed 5:1 with
NCI-N87 cells and treated for 18 h with (E1)-3s before measuring
CD69 expression by flow cytometry. Percent CD69-positive CD8.sup.+
T cells after incubation with 0.1 nM (E1)-3s (E) and/or 0.1 nM
peginterferonalfa-2a (P), in the absence or presence of NCI-N87
target cells (T). Each treatment was assayed in triplicate. Error
bars, S.D. *, P<0.001.
FIG. 29D. T-cell activation. Purified T cells were mixed 5:1 with
NCI-N87 cells and treated for 18 h with (E1)-3s before measuring
CD69 expression by flow cytometry. Geometric mean fluorescence of
the CD69.sup.+ cells after incubation with 0.1 nM (E1)-3s (E)
and/or 0.1 nM peginterferonalfa-2a (P), in the absence or presence
of NCI-N87 target cells (T). Each treatment was assayed in
triplicate. Error bars, S.D. *, P<0.001.
FIG. 30A. In-vivo efficacy with human pancreatic and gastric cancer
xenografts. Groups of 8 mice inoculated with human T cells and
Capan-1 pancreatic cancer cells were treated daily for five days
with 50 .mu.g of (E1)-3s (.tangle-solidup., solid black) or 60
.mu.g TF12 (, gray), once weekly for four weeks with 0.6 .mu.g of
peginterferonalfa-2a (*, solid black), a combination of (E1)-3s and
peginterferonalfa-2a regimens (.circle-solid., solid black) or with
saline (.circle-solid., dashed black). An additional group was
inoculated with Capan-1, but not T cells, and treated with
peginterferonalfa-2a (.quadrature., dashed black). Top panel,
Kaplan-Meyer survival plots. Bottom panel, mean tumor volumes
(+S.D.) vs days. Data marked with an asterisk were adapted from
FIG. 6C in Rossi et al. (2014, MAbs 6:381-91).
FIG. 30B. In-vivo efficacy with human pancreatic and gastric cancer
xenografts. Groups of 8 mice inoculated with human T cells and
Capan-1 pancreatic cancer cells were treated daily for five days
with 50 .mu.g of (E1)-3s (.tangle-solidup., solid black) or 60
.mu.g TF12 (, gray), once weekly for four weeks with 0.6 .mu.g of
peginterferonalfa-2a (*, solid black), a combination of (E1)-3s and
peginterferonalfa-2a regimens (.circle-solid., solid black) or with
saline (.circle-solid., dashed black). An additional group was
inoculated with Capan-1, but not T cells, and treated with
peginterferonalfa-2a (.quadrature., dashed black). Top panel,
Kaplan-Meyer survival plots. Bottom panel, mean tumor volumes
(+S.D.) vs days. Data marked with an asterisk were adapted from
FIG. 6C in Rossi et al. (2014, MAbs 6:381-91).
FIG. 30C. In-vivo efficacy with human pancreatic and gastric cancer
xenografts. Groups of 8 mice inoculated with NCI-N87 gastric cancer
cells were treated daily for five days with 50 .mu.g of (E1)-3s
(.tangle-solidup., solid black) or 60 .mu.g TF12 (, gray), once
weekly for four weeks with 0.6 .mu.g of peginterferonalfa-2a (*,
solid black), a combination of (E1)-3s and peginterferonalfa-2a
regimens (.circle-solid., solid black) or with saline
(.circle-solid., dashed black). An additional group was inoculated
with Capan-1, but not T cells, and treated with
peginterferonalfa-2a (.quadrature., dashed black). Top panel,
Kaplan-Meyer survival plots. Bottom panel, mean tumor volumes
(+S.D.) vs days. Data marked with an asterisk were adapted from
FIG. 6C in Rossi et al. (2014, MAbs 6:381-91).
FIG. 31. Cytokine production induced by E1-3. PBMCs were combined
at a 5:1 ratio with BxPC-3 cells and treated with a titration of
E1-3 for 24 h. Cytokine concentrations were measured using
Single-Analyte ELISArray kits (Qiagen). All cytokine levels were
<10 pg/mL in the absence of E1-3.
FIG. 32. In vitro redirected T cell killing of pancreatic and
gastric cancer cell lines. Purified CD8.sup.+ T cells
(1.2.times.10.sup.5/well) were mixed 6:1 with target cells
(2.times.10.sup.4/well) and treated with titrations of E1-3 in a
96-well plate. After 48 h, wells were washed to remove T cells and
the viable target cell densities were determined with an MTS assay.
Example of results for one of several T cell donors.
FIG. 33A. In vivo therapy of human gastric tumor xenografts. PBMCs
were mixed 2:1 with NCI-N87 cells and injected s.c. with matrigel
in NOD-SCID mice. Animals were given 50 .mu.g E1-3 i.v. on Days 0
and 3. Mice were monitored daily for signs of tumor out-growth,
after which tumors were measured twice weekly with an endpoint
measurement of >1.0 cm.sup.3. After 176 days, 7 of 8 mice in the
E1-3 treatment group had not reached the endpoint with 6 animals
remaining tumor free.
FIG. 33B. In vivo therapy of human gastric tumor xenografts. PBMCs
were mixed 2:1 with NCI-N87 cells and injected s.c. with matrigel
in NOD-SCID mice. Animals were given 50 .mu.g E1-3 i.v. on Days 0
and 3. Mice were monitored daily for signs of tumor out-growth,
after which tumors were measured twice weekly with an endpoint
measurement of >1.0 cm.sup.3. Tumors in the control group
comprising only PBMCs and NCI-87 reached the end point with a
median time of 39.5 days.
DETAILED DESCRIPTION
Definitions
Unless otherwise specified, "a" or "an" means "one or more".
As used herein, the terms "and" and "or" may be used to mean either
the conjunctive or disjunctive. That is, both terms should be
understood as equivalent to "and/or" unless otherwise stated.
A "therapeutic agent" is an atom, molecule, or compound that is
useful in the treatment of a disease. Examples of therapeutic
agents include antibodies, antibody fragments, peptides, drugs,
toxins, enzymes, nucleases, hormones, immunomodulators, antisense
oligonucleotides, small interfering RNA (siRNA), chelators, boron
compounds, photoactive agents, dyes, and radioisotopes.
An "antibody" as used herein refers to a full-length (i.e.,
naturally occurring or formed by normal immunoglobulin gene
fragment recombinatorial processes) immunoglobulin molecule (e.g.,
an IgG antibody) or an immunologically active (i.e., specifically
binding) portion of an immunoglobulin molecule, like an antibody
fragment. An "antibody" includes monoclonal, polyclonal,
bispecific, multispecific, murine, chimeric, humanized and human
antibodies.
A "naked antibody" is an antibody or antigen binding fragment
thereof that is not attached to a therapeutic or diagnostic agent.
The Fc portion of an intact naked antibody can provide effector
functions, such as complement fixation and ADCC (see, e.g.,
Markrides, Pharmacol Rev 50:59-87, 1998). Other mechanisms by which
naked antibodies induce cell death may include apoptosis. (Vaswani
and Hamilton, Ann Allergy Asthma Immunol 81: 105-119, 1998.)
An "antibody fragment" is a portion of an intact antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, scFv, dAb and the like.
Regardless of structure, an antibody fragment binds with the same
antigen that is recognized by the full-length antibody. For
example, antibody fragments include isolated fragments consisting
of the variable regions, such as the "Fv" fragments consisting of
the variable regions of the heavy and light chains or recombinant
single chain polypeptide molecules in which light and heavy
variable regions are connected by a peptide linker ("scFv
proteins"). "Single-chain antibodies", often abbreviated as "scFv"
consist of a polypeptide chain that comprises both a V.sub.H and a
V.sub.L domain which interact to form an antigen-binding site. The
V.sub.H and V.sub.L domains are usually linked by a peptide of 1 to
25 amino acid residues. Antibody fragments also include diabodies,
triabodies and single domain antibodies (dAb).
A "chimeric antibody" is a recombinant protein that contains the
variable domains including the complementarity determining regions
(CDRs) of an antibody derived from one species, preferably a rodent
antibody, while the constant domains of the antibody molecule are
derived from those of a human antibody. For veterinary
applications, the constant domains of the chimeric antibody may be
derived from that of other species, such as a cat or dog.
A "humanized antibody" is a recombinant protein in which the CDRs
from an antibody from one species; e.g., a rodent antibody, are
transferred from the heavy and light variable chains of the rodent
antibody into human heavy and light variable domains, including
human framework region (FR) sequences. The constant domains of the
antibody molecule are derived from those of a human antibody. To
maintain binding activity, a limited number of FR amino acid
residues from the parent (e.g., murine) antibody may be substituted
for the corresponding human FR residues.
A "human antibody" is an antibody obtained from transgenic mice
that have been genetically engineered to produce specific human
antibodies in response to antigenic challenge. In this technique,
elements of the human heavy and light chain locus are introduced
into strains of mice derived from embryonic stem cell lines that
contain targeted disruptions of the endogenous heavy chain and
light chain loci. The transgenic mice can synthesize human
antibodies specific for human antigens, and the mice can be used to
produce human antibody-secreting hybridomas. Methods for obtaining
human antibodies from transgenic mice are described by Green et
al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856
(1994), and Taylor et al., Int. Immun. 6:579 (1994). A human
antibody also can be constructed by genetic or chromosomal
transfection methods, as well as phage display technology, all of
which are known in the art. (See, e.g., McCafferty et al., 1990,
Nature 348:552-553 for the production of human antibodies and
fragments thereof in vitro, from immunoglobulin variable domain
gene repertoires from unimmunized donors). In this technique,
antibody variable domain genes are cloned in-frame into either a
major or minor coat protein gene of a filamentous bacteriophage,
and displayed as functional antibody fragments on the surface of
the phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties. In
this way, the phage mimics some of the properties of the B cell.
Phage display can be performed in a variety of formats, for their
review, see, e.g. Johnson and Chiswell, Current Opinion in
Structural Biology 3:5564-571 (1993). Human antibodies may also be
generated by in vitro activated B cells. (See, U.S. Pat. Nos.
5,567,610 and 5,229,275).
As used herein, the term "antibody fusion protein" is a
recombinantly produced antigen-binding molecule in which an
antibody or antibody fragment is linked to another protein or
peptide, such as the same or different antibody or antibody
fragment or a DDD or AD peptide. The fusion protein may comprise a
single antibody component, a multivalent or multispecific
combination of different antibody components or multiple copies of
the same antibody component. The fusion protein may additionally
comprise an antibody or an antibody fragment and a therapeutic
agent. Examples of therapeutic agents suitable for such fusion
proteins include immunomodulators and toxins. One preferred toxin
comprises a ribonuclease (RNase), preferably a recombinant RNase. A
preferred immunomodulator might be an interferon, such as
interferon-.alpha., interferon-.beta. or interferon-.lamda..
A "multispecific antibody" is an antibody that can bind
simultaneously to at least two targets that are of different
structure, e.g., two different antigens, two different epitopes on
the same antigen, or a hapten and/or an antigen or epitope. A
"multivalent antibody" is an antibody that can bind simultaneously
to at least two targets that are of the same or different
structure. Valency indicates how many binding arms or sites the
antibody has to a single antigen or epitope; i.e., monovalent,
bivalent, trivalent or multivalent. The multivalency of the
antibody means that it can take advantage of multiple interactions
in binding to an antigen, thus increasing the avidity of binding to
the antigen. Specificity indicates how many antigens or epitopes an
antibody is able to bind; i.e., monospecific, bispecific,
trispecific, multispecific. Using these definitions, a natural
antibody, e.g., an IgG, is bivalent because it has two binding arms
but is monospecific because it binds to one epitope. Multispecific,
multivalent antibodies are constructs that have more than one
binding site of different specificity.
A "bispecific antibody" is an antibody that can bind simultaneously
to two targets which are of different structure. Bispecific
antibodies (bsAb) and bispecific antibody fragments (bsFab) may
have at least one arm that specifically binds to, for example, a T
cell, an NK cell, a monocyte or a neutrophil, and at least one
other arm that specifically binds to an antigen produced by or
associated with a diseased cell, tissue, organ or pathogen, for
example a tumor-associated antigen. A variety of bispecific
antibodies can be produced using molecular engineering.
An antibody preparation, or a composition described herein, is said
to be administered in a "therapeutically effective amount" if the
amount administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient subject. In particular
embodiments, an antibody preparation is physiologically significant
if its presence invokes an antitumor response or mitigates the
signs and symptoms of an infectious disease state. A
physiologically significant effect could also be the evocation of a
humoral and/or cellular immune response in the recipient subject
leading to growth inhibition or death of target cells.
Anti-Trop-2 Antibodies
In preferred embodiments, the subject bispecific antibody includes
at least one antibody or fragment thereof that binds to Trop-2. In
a more preferred embodiment, the anti-Trop-2 antibody may is a
humanized RS7 antibody (see, e.g., U.S. Pat. No. 7,238,785,
incorporated herein by reference in its entirety), comprising the
light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:115); CDR2
(SASYRYT, SEQ ID NO:116); and CDR3 (QQHYITPLT, SEQ ID NO:117) and
the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:118); CDR2
(WINTYTGEPTYTDDFKG, SEQ ID NO:119) and CDR3 (GGFGSSYWYFDV, SEQ ID
NO:120).
The RS7 antibody was a murine IgG.sub.1 raised against a crude
membrane preparation of a human primary squamous cell lung
carcinoma. (Stein et al., Cancer Res. 50: 1330, 1990) The RS7
antibody recognizes a 46-48 kDa glycoprotein, characterized as
cluster 13. (Stein et al., Int. J. Cancer Supp. 8:98-102, 1994) The
antigen was designated as EGP-1 (epithelial glycoprotein-1), but is
also referred to as Trop-2.
Trop-2 is a type-I transmembrane protein and has been cloned from
both human (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse
cells (Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to
its role as a tumor-associated calcium signal transducer (Ripani et
al., Int J Cancer 1998; 76:671-6), the expression of human Trop-2
was shown to be necessary for tumorigenesis and invasiveness of
colon cancer cells, which could be effectively reduced with a
polyclonal antibody against the extracellular domain of Trop-2
(Wang et al., Mol Cancer Ther 2008; 7:280-5).
The growing interest in Trop-2 as a therapeutic target for solid
cancers (Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is
attested by further reports that documented the clinical
significance of overexpressed Trop-2 in breast (Huang et al., Clin
Cancer Res 2005; 11:4357-64), colorectal (Ohmachi et al., Clin
Cancer Res 2006; 12:3057-63; Fang et al., Int J Colorectal Dis
2009; 24:875-84), and oral squamous cell (Fong et al., Modern
Pathol 2008; 21:186-91) carcinomas. The latest evidence that
prostate basal cells expressing high levels of Trop-2 are enriched
for in vitro and in vivo stem-like activity is particularly
noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;
105:20882-7).
Flow cytometry and immunohistochemical staining studies have shown
that the RS7 MAb detects antigen on a variety of tumor types, with
limited binding to normal human tissue (Stein et al., 1990). Trop-2
is expressed primarily by carcinomas such as carcinomas of the
lung, stomach, urinary bladder, breast, ovary, uterus, and
prostate. Localization and therapy studies using radiolabeled
murine RS7 MAb in animal models have demonstrated tumor targeting
and therapeutic efficacy (Stein et al., 1990; Stein et al.,
1991).
Strong RS7 staining has been demonstrated in tumors from the lung,
breast, bladder, ovary, uterus, stomach, and prostate. (Stein et
al., Int. J. Cancer 55:938, 1993) The lung cancer cases comprised
both squamous cell carcinomas and adenocarcinomas. (Stein et al.,
Int. J. Cancer 55:938, 1993) Both cell types stained strongly,
indicating that the RS7 antibody does not distinguish between
histologic classes of non-small-cell carcinoma of the lung.
The RS7 MAb is rapidly internalized into target cells (Stein et
al., 1993). The internalization rate constant for RS7 MAb is
intermediate between the internalization rate constants of two
other rapidly internalizing MAbs, which have been demonstrated to
be useful for immunotoxin production. (Id.) It is well documented
that internalization of immunotoxin conjugates is a requirement for
anti-tumor activity. (Pastan et al., Cell 47:641, 1986)
Internalization of drug immunoconjugates has been described as a
major factor in anti-tumor efficacy. (Yang et al., Proc. Nat'l
Acad. Sci. USA 85: 1189, 1988) Thus, the RS7 antibody exhibits
several important properties for therapeutic applications.
While the hRS7 antibody is preferred, other anti-Trop-2 antibodies
are known and/or publicly available and in alternative embodiments
may be utilized in the subject ADCs. While humanized or human
antibodies are preferred for reduced immunogenicity, in alternative
embodiments a chimeric antibody may be of use. As discussed below,
methods of antibody humanization are well known in the art and may
be utilized to convert an available murine or chimeric antibody
into a humanized form.
Anti-Trop-2 antibodies are commercially available from a number of
sources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417
(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01,
10428-MM02, 10428-R001, 10428-R030 (Sino Biological Inc., Beijing,
China); MR54 (eBioscience, San Diego, Calif.); sc-376181,
sc-376746, Santa Cruz Biotechnology (Santa Cruz, Calif.);
MM0588-49D6, (Novus Biologicals, Littleton, Colo.); ab79976, and
ab89928 (ABCAM.RTM., Cambridge, Mass.).
Other anti-Trop-2 antibodies have been disclosed in the patent
literature. For example, U.S. Publ. No. 2013/0089872 discloses
anti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107
(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM
BP-11253), T6-16 (Accession No. FERM BP-11346), and T5-86
(Accession No. FERM BP-11254), deposited with the International
Patent Organism Depositary, Tsukuba, Japan. U.S. Pat. No. 5,840,854
disclosed the anti-Trop-2 monoclonal antibody BR110 (ATCC No.
HB11698). U.S. Pat. No. 7,420,040 disclosed an anti-Trop-2 antibody
produced by hybridoma cell line AR47A6.4.2, deposited with the IDAC
(International Depository Authority of Canada, Winnipeg, Canada) as
accession number 141205-05. U.S. Pat. No. 7,420,041 disclosed an
anti-Trop-2 antibody produced by hybridoma cell line AR52A301.5,
deposited with the IDAC as accession number 141205-03. U.S. Publ.
No. 2013/0122020 disclosed anti-Trop-2 antibodies 3E9, 6G11, 7E6,
15E2, 18B1. Hybridomas encoding a representative antibody were
deposited with the American Type Culture Collection (ATCC),
Accession Nos. PTA-12871 and PTA-12872. U.S. Pat. No. 8,715,662
discloses anti-Trop-2 antibodies produced by hybridomas deposited
at the AID-ICLC (Genoa, Italy) with deposit numbers PD 08019, PD
08020 and PD 08021. U.S. Patent Application Publ. No. 20120237518
discloses anti-Trop-2 antibodies 77220, KM4097 and KM4590. U.S.
Pat. No. 8,309,094 (Wyeth) discloses antibodies A1 and A3,
identified by sequence listing. The Examples section of each patent
or patent application cited above in this paragraph is incorporated
herein by reference. Non-patent publication Lipinski et al. (1981,
Proc Natl. Acad Sci USA, 78:5147-50) disclosed anti-Trop-2
antibodies 162-25.3 and 162-46.2.
Numerous anti-Trop-2 antibodies are known in the art and/or
publicly available. As discussed below, methods for preparing
antibodies against known antigens were routine in the art. The
sequence of the human Trop-2 protein was also known in the art
(see, e.g., GenBank Accession No. CAA54801.1). Methods for
producing humanized, human or chimeric antibodies were also known.
The person of ordinary skill, reading the instant disclosure in
light of general knowledge in the art, would have been able to make
and use the genus of anti-Trop-2 antibodies in the subject
ADCs.
Anti-CD3 Antibodies
A variety of antibodies against CD3 that may be used in the claimed
methods and compositions are publicly known and/or commercially
available, such as from LSBio (catalog Nos. LS-B6698, LS-B8669;
LS-B8765, LS-C96311, LS-C58677, etc.); ABCAM.RTM. (catalog Nos.
ab5690, ab16669, ab699, ab828, ab8671, etc.); Santa Cruz
Biotechnology (catalog Nos. sc-20047, sc-20080, sc-19590, sc-59008,
sc-101442, etc.); and many other suppliers.
In a preferred embodiment, the amino acid sequence of the anti-CD3
moiety, used as part of a DNL.TM. complex, is as disclosed below in
SEQ ID NO:96 to SEQ ID NO:101. However, the person of ordinary
skill will realize that any known anti-CD3 antibody may be utilized
in the claimed methods and compositions. Preferably, the antibody
moieties of use are humanized or human.
Leukocyte Redirecting Bispecific Antibody Complexes
In preferred embodiments, the subject bispecific antibodies
comprise an anti-CD3.times.anti-Trop-2 antibody. As discussed
above, various antibodies against CD3 or Trop-2 are known in the
art and any such known antibody may be utilized. However, in
alternative embodiments, antibodies against other leukocyte
antigens than CD3 or against other disease-associated antigens than
Trop-2 may be utilized.
Exemplary T-cell antigens include CD2, CD3, CD4, CD5, CD6, CD8,
CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90. Other
exemplary antigens may be selected from CD8, CD16, CD56, CD57,
ADAM17, and CD137 for NK cells; CD74, HLA-DR alpha chain, CD14,
CD16, CD64 and CD89 for monocytes; and CEACAM6, CEACAM8, CD16b,
CD32a, CD89, CD177, CD11a, CD11b and SLC44A2 for neutrophils.
Preferably, the anti-T-cell antibody binds to CD3, or the anti-NK
antibody binds to CD16. As discussed below, many examples of
disease-associated antigens, such as tumor-associated antigens
(TAAs) are known. An exemplary preferred TAA is Trop-2.
Certain alternative embodiments may concern an
anti-CD3.times.anti-CD19 bispecific antibody. Various bispecific
anti-CD3.times.anti-CD19 antibodies are known in the art and
presently in clinical development, such as BITE.RTM. (Bispecific
T-cell Engager) (e.g., Nagorsen et al., 2009, Leukemia &
Lymphoma 50:886-91; Amann et al., 2009, J Immunother 32:453-64;
Baeuerle and Reinhardt, 2009, Cancer Res 69:4941-44) and DART.RTM.
(see, e.g., Moore et al., 2011, Blood 117:4542-51; Veri et al.,
2010, Arthritis Rheum 62:1933-43). Blinatumomab is a BITE.RTM.
antibody comprising V.sub.H and V.sub.L domains of anti-CD3 and
anti-CD19 antibody fragments, connected with a 5-amino acid linker
and expressed as a single polypeptide chain that anneals to itself
to form the antigen-binding sites. It is thought that blinatumomab
acts by bringing the T-cell-specific CD3 and B-cell specific CD19
antigens into close proximity, to initiate a T-cell cytotoxic
response against the juxtaposed B cells, which does not require
T-cell specificity to the cancer cells (e.g., Portell et al., 2013,
Clin Pharmacol 5(Suppl 1): 5-11). Due to its short half-life,
blinatumomab requires continuous intravenous infusion to be
effective, (Portell et al., 2013). A phase II trial of B-cell ALL
patients with persistent or relapsed minimal residual disease
reported an approximately 80% rate of complete remission (Portell
et al., 2013).
Doses of blinatumomab as low as 0.005 mg/m.sup.2/day were reported
to be effective to eliminate cancer cells in non-Hodgkin's lymphoma
patients (Bargou et al., 2008, Science 321:974-77). Partial and
complete remissions were observed starting at a dose level of 0.015
mg and all six patients tested at a dose of 0.06 mg experienced a
tumor regression (Bargou et al., 2008). In vitro, blinatumomab
induced 50% cell lysis of MEC-1 cells at a concentration of 10
pg/mL (Topp et al., 2012, Blood 120:5185-87; Bassan et al., 2012,
Blood 120:5094-95).
The anti-CD19 portion of blinatumomab was derived from the HD37
hybridoma (see, e.g., U.S. Pat. No. 7,575,923, the Examples section
of which is incorporated herein by reference), which is publicly
available (e.g., Santa Cruz Biotechnology Cat. No. sc-18894). The
anti-CD3 portion of blinatumomab was derived from the TR66
hybridoma (U.S. Pat. No. 7,575,923; Traunecker et al., 1991, EMBO
J. 10:3655-59), also publicly available (e.g., Enzo Life Sciences,
catalog No. ALX-804-822-C100).
A variety of antibodies against CD19 are publicly known and/or
commercially available, such as from Santa Cruz Biotechnology
(catalog Nos. sc-390244, sc-373897, sc-18894, sc-18896, etc.);
ABCAM.RTM. (catalog Nos. ab25232, ab134114, ab140981, ab1255,
etc.); ABBIOTEC.TM. (catalog Nos. 252262, 252248, 250585, 251063,
etc.) and many other vendors.
In a preferred embodiment, the anti-CD19 antibody moiety is a
humanized A19 antibody, comprising the light chain CDR sequences
CDR1 KASQSVDYDGDSYLN (SEQ ID NO:90); CDR2 DASNLVS (SEQ ID NO:91);
and CDR3 QQSTEDPWT (SEQ ID NO:92) and the heavy chain CDR sequences
CDR1 SYWMN (SEQ ID NO:93); CDR2 QIWPGDGDTNYNGKFKG (SEQ ID NO:94)
and CDR3 RETTTVGRYYYAMDY (SEQ ID NO:95).
Other anti-CD3.times.anti-CD19 bispecific antibodies are known,
such as DART.RTM., which also incorporates the anti-CD19 Fv
sequences of HD37 and the anti-CD3 Fv sequences of TR66 (Moore et
al., 2011, Blood 117:4542-51; Veri et al., 2010, Arthritis Rheum
62:1933-43). Moore et al. (2011) reported that DART.RTM. bispecific
antibodies were more potent at inducing B cell lysis than
single-chain, bispecific antibodies (BITE.RTM.) bearing identical
anti-CD19 and anti-CD3 variable region sequences, with EC.sub.50
values in the pg/mL range (Moore et al., 2011). Other
anti-CD3.times.anti-CD19 bispecific antibodies besides DART.RTM.
and BITE.RTM. have been reported (see, e.g., Wei et al., 2012, Cell
Oncol 35:423-34; Portner et al., 2012, Cancer Immunol Immunother
61:1869-75; Zhou et al., 2012, Biotechnol Lett. 34:1183-91). In
certain embodiments, any known anti-CD3.times.anti-CD19 bispecific
antibody may be used to induce an immune response against
disease-associated cells.
Catumaxomab is an anti-CD3.times.anti-EpCAM bispecific antibody
that has been approved in Europe for treatment of malignant ascites
associated with metastasizing cancer (Chames & Baty, 2009, MAbs
1:539-47). In a mouse model system, catumaxomab was able to kill
tumor cells at a concentration range of 10 pM and was reported to
lead to total eradication of melanoma tumors (Chames & Baty,
2009). Human clinical trials with ovarian cancer patients with
malignant ascites also showed a statistically significant efficacy
(Chames & Baty, 2009). However, the high immunogenicity of the
rat/mouse hybrid bsAb may limit i.v. administration of the antibody
(Chames & Baty, 2009). The use of anti-tumor bsAbs is not
limited to anti-CD3.times.anti-CD19, but has also included
anti-HER2.times.anti-CD64 (MDX-210, MDX-H210),
anti-EGFR.times.anti-CD64 (MDX-447), anti-CD30.times.anti-CD16
(HRS-3/A9), anti-HER2.times.anti-CD3 (Her2Bi),
anti-CD20.times.anti-CD3 (CD20Bi, Bi20), anti-EpCAM.times.anti-CD3
(catumaxomab, MT110), anti-HER2.times.anti-CD3 (ertumaxomab), and
anti-NG2.times.anti-CD28 (rM28) (Chames & Baty, 2009).
The person of ordinary skill will realize that the subject
leukocyte redirecting bispecific antibodies are not limited to
anti-CD3.times.anti-Trop-2 constructs, but may comprise antibodies
against any known disease-associated antigens attached to an
anti-CD3 antibody moiety. Alternatively, antibodies against other
T-cell antigens besides CD3, or other antigens expressed on NK
cells, monocytes or neutrophils may also be used. Exemplary T-cell
antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD6,
CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90.
Other exemplary antigens may be selected from CD8, CD16, CD56,
CD57, ADAM17, KIR and CD137 for NK cells; CD74, HLA-DR alpha chain,
CD14, CD16, CD64 and CD89 for monocytes; and CEACAM6, CEACAM8,
CD16b, CD32a, CD89, CD177, CD11a, CD11b and SLC44A2 for
neutrophils. Antibodies against each of the leukocyte antigens are
publicly known and/or publicly available (see, e.g., ABCAM.RTM.
catalog numbers ab131276, ab139266, ab8360, ab51312, ab846,
ab133616, ab75877, ab133255, ab109217, ab93278, ab17147, ab115851,
ab128955, ab13463, ab85986; Santa Cruz Biotechnology catalog
numbers sc-46683, sc-59047; Enzo Life Sciences, Inc. catalog number
ALX-805-037-C100; Sino Biological Inc. catalog numbers 12211-RP02,
11150-R074; Millipore catalog numbers 04-1102, 04-1102, MAB1406).
These and numerous other anti-leukocyte antibodies were publicly
available and could have been used in the subject leukocyte
redirecting bsAbs. As discussed below, numerous antibodies against
a wide variety of disease-associated antigens were publicly known
and/or commercially available and could have been used in the
subject leukocyte redirecting bispecific antibodies. Other
exemplary leukocyte redirecting bsAbs of potential use include
FBTA05 (anti-CD20.times.anti-CD3) and TRBS07
(anti-GD2.times.anti-CD3).
Interferon Therapy
In various embodiments, the subject bispecific antibodies may be
used in combination with one or more interferons, such as
interferon-.alpha., interferon-.beta. or interferon-.lamda.,
preferably interferon-.alpha.. Human interferons are well known in
the art and the amino acid sequences of human interferons may be
readily obtained from public databases (e.g., GenBank Accession
Nos. AAA52716.1; AAA52724; AAC41702.1; EAW56871.1; EAW56870.1;
EAW56869.1). Human interferons may also be commercially obtained
from a variety of vendors (e.g., Cell Signaling Technology, Inc.,
Danvers, Mass.; Genentech, South San Francisco, Calif.; EMD
Millipore, Billerica, Mass.).
Interferon-.alpha. (IFN.alpha.) has been reported to have
anti-tumor activity in animal models of cancer (Ferrantini et al.,
1994, J Immunol 153:4604-15) and human cancer patients (Gutterman
et al., 1980, Ann Intern Med 93:399-406). IFN.alpha. can exert a
variety of direct anti-tumor effects, including down-regulation of
oncogenes, up-regulation of tumor suppressors, enhancement of
immune recognition via increased expression of tumor surface MHC
class I proteins, potentiation of apoptosis, and sensitization to
chemotherapeutic agents (Gutterman et al., 1994, PNAS USA
91:1198-205; Matarrese et al., 2002, Am J Pathol 160:1507-20;
Mecchia et al., 2000, Gene Ther 7:167-79; Sabaawy et al., 1999, Int
J Oncol 14:1143-51; Takaoka et al, 2003, Nature 424:516-23). For
some tumors, IFN.alpha. can have a direct and potent
anti-proliferative effect through activation of STAT1 (Grimley et
al., 1998 Blood 91:3017-27). Interferon-.alpha.2b has been
conjugated to anti-tumor antibodies, such as the hL243 anti-HLA-DR
antibody and depletes lymphoma and myeloma cells in vitro and in
vivo (Rossi et al., 2011, Blood 118:1877-84).
Indirectly, IFN.alpha. can inhibit angiogenesis (Sidky and Borden,
1987, Cancer Res 47:5155-61) and stimulate host immune cells, which
may be vital to the overall antitumor response but has been largely
under-appreciated (Belardelli et al., 1996, Immunol Today
17:369-72). IFN.alpha. has a pleiotropic influence on immune
responses through effects on myeloid cells (Raefsky et al, 1985, J
Immunol 135:2507-12; Luft et al, 1998, J Immunol 161:1947-53),
T-cells (Carrero et al, 2006, J Exp Med 203:933-40; Pilling et al.,
1999, Eur J Immunol 29:1041-50), and B-cells (Le et al, 2001,
Immunity 14:461-70). As an important modulator of the innate immune
system, IFN.alpha. induces the rapid differentiation and activation
of dendritic cells (Belardelli et al, 2004, Cancer Res 64:6827-30;
Paquette et al., 1998, J Leukoc Biol 64:358-67; Santini et al.,
2000, J Exp Med 191:1777-88) and enhances the cytotoxicity,
migration, cytokine production and antibody-dependent cellular
cytotoxicity (ADCC) of NK cells (Biron et al., 1999, Ann Rev
Immunol 17:189-220; Brunda et al. 1984, Cancer Res 44:597-601).
Interferon-.beta. has been reported to be efficacious for therapy
of a variety of solid tumors. Patients treated with 6 million units
of IFN-.beta. twice a week for 36 months showed a decreased
recurrence of hepatocellular carcinoma after complete resection or
ablation of the primary tumor in patients with HCV-related liver
cancer (Ikeda et al., 2000, Hepatology 32:228-32). Gene therapy
with interferon-.beta. induced apoptosis of glioma, melanoma and
renal cell carcinoma (Yoshida et al., 2004, Cancer Sci 95:858-65).
Endogenous IFN-.beta. has been observed to inhibit tumor growth by
inhibiting angiogenesis in vivo (Jablonska et al., 2010, J Clin
Invest. 120:1151-64.)
IFN-.lamda.s, designated as type III interferons, are a newly
described group of cytokines that consist of IFN-.lamda.1, 2, 3
(also referred to as interleukin-29, 28A, and 28B, respectively),
that are genetically encoded by three different genes located on
chromosome 19 (Kotenko et al., 2003, Nat Immunol 4:69-77; Sheppard
et al., 2003, Nat Immunol 4:63-8). At the protein level,
IFN-.lamda.2 and -.lamda.3 are is highly homologous, with 96% amino
acid identity, while IFN-.lamda.1 shares approximately 81% homology
with IFN-.lamda.2 and -.lamda.3 (Sheppard et al., 2003, Nat Immunol
4:63-8). IFN-.lamda.s activate signal transduction via the JAK/STAT
pathway similar to that induced by type I IFN, including the
activation of JAK1 and TYK2 kinases, the phosphorylation of STAT
proteins, and the activation of the transcription complex of
IFN-stimulated gene factor 3 (ISGF3) (Witte et al., 2010, Cytokine
Growth Factor Rev 21:237-51; Zhou et al., 2007, J Virol
81:7749-58).
A major difference between type III and type I IFN systems is the
distribution of their respective receptor complexes.
IFN-.alpha./.beta. signals through two extensively expressed type I
interferon receptors, and the resulting systemic toxicity
associated with IFN-.alpha./.beta. administration has limited their
use as therapeutic agents (Pestka et al., 2007, J Biol Chem
282:20047-51). In contrast, IFN-.lamda.s signal through a
heterodimeric receptor complex consisting of unique IFN-.lamda.,
receptor 1 (IFN-.lamda.R1) and IL-10 receptor 2 (IL-10R2). As
previously reported (Witte et al., 2009, Genes Immun 10:702-14),
IFN-.lamda.R1 has a very restricted expression pattern with the
highest levels in epithelial cells, melanocytes, and hepatocytes,
and the lowest level in primary central nervous system (CNS) cells.
Blood immune system cells express high levels of a short
IFN-.lamda. receptor splice variant (sIFN-.lamda.R1) that inhibits
IFN-.lamda. action. The limited responsiveness of neuronal cells
and immune cells implies that the severe toxicity frequently
associated with IFN-.alpha. therapy may be absent or significantly
reduced with IFN-.lamda.s (Witte et al., 2009, Genes Immun
10:702-14; Witte et al., 2010, Cytokine Growth Factor Rev
21:237-51). A recent publication reported that while IFN-.alpha.
and IFN-.lamda. induce expression of a common set of ISGs
(interferon-stimulated genes) in hepatocytes, unlike IFN-.alpha.,
administration of IFN-.lamda. did not induce STAT activation or ISG
expression in purified lymphocytes or monocytes (Dickensheets et
al., 2013, J Leukoc Biol. 93, published online Dec. 20, 2012). It
was suggested that IFN-.lamda. may be superior to IFN-.alpha. for
treatment of chronic HCV infection, as it is less likely to induce
leukopenias that are often associated with IFN-.alpha. therapy
(Dickensheets et al., 2013).
IFN-.lamda.s display structural features similar to IL-10-related
cytokines, but functionally possess type I IFN-like anti-viral and
anti-proliferative activity (Witte et al., 2009, Genes Immun
10:702-14; Ank et al., 2006, J Virol 80:4501-9; Robek et al., 2005,
J Virol 79:3851-4). IFN-.lamda.1 and -.lamda.2 have been
demonstrated to reduce viral replication or the cytopathic effect
of various viruses, including DNA viruses (hepatitis B virus (Robek
et al., 2005, J Virol 79:3851-4, Doyle et al., 2006, Hepatology
44:896-906) and herpes simplex virus 2 (Ank et al., 2008, J Immunol
180:2474-85)), ss (+) RNA viruses (EMCV; Sheppard et al., 2003, Nat
Immunol 4:63-8) and hepatitis C virus (Robek et al., 2005, J Virol
79:3851-4, Doyle et al., 2006, Hepatology 44:896-906; Marcello et
al., 2006, Gastroenterol 131:1887-98; Pagliaccetti et al., 2008, J
Biol Chem 283:30079-89), ss (-) RNA viruses (vesicular stomatitis
virus; Pagliaccetti et al., 2008, J Biol Chem 283:30079-89) and
influenza-A virus (Jewell et al., 2010, J Virol 84:11515-22) and
double-stranded RNA viruses, such as rotavirus (Pott et al., 2011,
PNAS USA 108:7944049). IFN-.lamda.3 has been identified from
genetic studies as a key cytokine in HCV infection (Ge et al.,
2009, Nature 461:399-401), and has also shown potent activity
against EMCV (Dellgren et al., 2009, Genes Immun 10:125-31). A
deficiency of rhinovirus-induced IFN-.lamda. production was
reported to be highly correlated with the severity of
rhinovirus-induced asthma exacerbation (Contoli et al., 2006,
Nature Med 12:1023-26) and IFN-.lamda. therapy has been suggested
as a new approach for treatment of allergic asthma (Edwards and
Johnston, 2011, EMBO Mol Med 3:306-8; Koltsida et al., 2011, EMBO
Mot Med 3:348-61).
The anti-proliferative activity of IFN-.lamda.s has been
established in several human cancer cell lines, including
neuroendocrine carcinoma BON1 (Zitzmann et al., 2006, Biochem
Biophys Res Commun 344:1334-41), glioblastoma LN319 (Meager et al.,
2005, Cytokine 31:109-18), immortalized keratinocyte HaCaT (Maher
et al., 2008, Cancer Biol Ther 7:1109-15), melanoma F01
(Guenterberg et al., 2010, Mol Cancer Ther 9:510-20), and
esophageal carcinoma TE-11 (Li et al., 2010, Eur J Cancer
46:180-90). In animal models, IFN-.lamda.s induce both tumor
apoptosis and destruction through innate and adaptive immune
responses, suggesting that local delivery of IFN-.lamda. might be a
useful adjunctive strategy in the treatment of human malignancies
(Numasaki et al., 2007, J Immunol 178:5086-98). A Fab-linked
interferon-.lamda. was demonstrated to have potent anti-tumor and
anti-viral activity in targeted cells (Liu et al., 2013, PLoS One
8:e63940).
In clinical settings, PEGylated IFN-.lamda.1 (PEG-IFN-.lamda.1) has
been provisionally used for patients with chronic hepatitis C virus
infection. In a phase Ib study (n=56), antiviral activity was
observed at all dose levels (0.5-3.0 .mu.g/kg), and viral load
reduced 2.3 to 4.0 logs when PEG-IFN-.lamda.1 was administrated to
genotype 1 HCV patients who relapsed after IFN-.alpha. therapy
(Muir et al., 2010, Hepatology 52:822-32). A phase IIb study
(n=526) showed that patients with HCV genotypes 1 and 4 had
significantly higher response rates to treatment with
PEG-IFN-.lamda.1 compared to PEG-IFN-.alpha.. At the same time,
rates of adverse events commonly associated with type I interferon
treatment were lower with PEG-IFN-.lamda.1 than with
PEG-IFN-.alpha.. Neutropenia and thrombocytopenia were infrequently
observed and the rates of flu-like symptoms, anemia, and
musculoskeletal symptoms decreased to about 1/3 of that seen with
PEG-IFN-.alpha. treatment. However, rates of serious adverse
events, depression and other common adverse events (.gtoreq.10%)
were similar between PEG-IFN-.lamda.1 and PEG-IFN-.alpha.. Higher
rates of hepatotoxicity were seen in the highest-dose
PEG-IFN-.lamda.1 compared with PEG-IFN-.alpha. ("Investigational
Compound PEG-Interferon Lambda Achieved Higher Response Rates with
Fewer Flu-like and Musculoskeletal Symptoms and Cytopenias Than
PEG-Interferon Alfa in Phase IIb Study of 526 Treatment-Naive
Hepatitis C Patients," Apr. 2, 2011, Press Release from
Bristol-Myers Squibb).
In various embodiments, the subject leukocyte redirecting
bispecific antibodies, ADCs and/or checkpoint inhibitor mAbs may be
used in combination with one or more interferons, such as
interferon-.alpha., interferon-.beta., interferon-.lamda.1,
interferon-.lamda.2, or interferon-.lamda.3. When used with other
agents, the interferon may be administered prior to, concurrently
with, or after the other agent. When administered concurrently, the
interferon may be either conjugated to or separate from the other
agent.
Checkpoint Inhibitor Antibodies
Studies with checkpoint inhibitor antibodies for cancer therapy
have generated unprecedented response rates in cancers previously
thought to be resistant to cancer treatment (see, e.g., Ott &
Bhardwaj, 2013, Frontiers in Immunology 4:346; Menzies & Long,
2013, Ther Adv Med Oncol 5:278-85; Pardoll, 2012, Nature Reviews
Cancer 12:252-64; Mavilio & Lugli). Therapy with antagonistic
checkpoint blocking antibodies against immune system checkpoints
such as CTLA4, PD1 and PD-L1 are one of the most promising new
avenues of immunotherapy for cancer and other diseases. In contrast
to the majority of anti-cancer agents, checkpoint inhibitors do not
target tumor cells directly, but rather target lymphocyte receptors
or their ligands in order to enhance the endogenous antitumor
activity of the immune system. (Pardoll, 2012, Nature Reviews
Cancer 12:252-264) Because such antibodies act primarily by
regulating the immune response to diseased cells, tissues or
pathogens, they may be used in combination with other therapeutic
modalities, such as the subject leukocyte redirecting bispecific
antibodies, ADCs and/or interferons to enhance the anti-tumor
effect of such agents.
It is now clear that tumors can escape immune surveillance by
co-opting certain immune-checkpoint pathways, particularly in T
cells that are specific for tumor antigens (Pardoll, 2012, Nature
Reviews Cancer 12:252-264). Because many such immune checkpoints
are initiated by ligand-receptor interactions, they can be readily
blocked by antibodies against the ligands and/or their receptors
(Pardoll, 2012, Nature Reviews Cancer 12:252-264). Although
checkpoint inhibitor antibodies against CTLA4, PD1 and PD-L1 are
the most clinically advanced, other potential checkpoint antigens
are known and may be used as the target of therapeutic antibodies,
such as LAG3, B7-H3, B7-H4 and TIM3 (Pardoll, 2012, Nature Reviews
Cancer 12:252-264).
Programmed cell death protein 1 (PD1, also known as CD279) encodes
a cell surface membrane protein of the immunoglobulin superfamily,
which is expressed in B cells and NK cells (Shinohara et al., 1995,
Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother
56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll, 2012,
Nature Reviews Cancer 12:252-264). The major role of PD1 is to
limit the activity of T cells in peripheral tissues during
inflammation in response to infection, as well as to limit
autoimmunity (Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1
expression is induced in activated T cells and binding of PD1 to
one of its endogenous ligants acts to inhibit T-cell activation by
inhibiting stimulatory kinases (Pardoll, 2012, Nature Reviews
Cancer 12:252-264). PD1 also acts to inhibit the TCR "stop signal"
(Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1 is highly
expressed on T.sub.reg cells and may increase their proliferation
in the presence of ligand (Pardoll, 2012, Nature Reviews Cancer
12:252-264).
Anti-PD1 antibodies have been used for treatment of melanoma,
non-small-cell lung cancer, bladder cancer, prostate cancer,
colorectal cancer, head and neck cancer, triple-negative breast
cancer, leukemia, lymphoma and renal cell cancer (Topalian et al.,
2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer
Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51;
Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96; Menzies &
Long, 2013, Ther Adv Med Oncol 5:278-85). Because PD1/PD-L1 and
CTLA4 act by different pathways, it is possible that combination
therapy with checkpoint inhibitor antibodies against each may
provide an enhanced immune response.
Exemplary anti-PD1 antibodies include lambrolizumab (MK-3475,
MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224
(MERCK), and pidilizumab (CT-011, CURETECH LTD.). Anti-PD1
antibodies are commercially available, for example from ABCAM.RTM.
(AB137132), BIOLEGEND.RTM. (EH12.2H7, RMP1-14) and AFFYMETRIX
EBIOSCIENCE (J105, J116, MIH4).
Programmed cell death 1 ligand 1 (PD-L1, also known as CD274 and
B7-H1) is a ligand for PD1, found on activated T cells, B cells,
myeloid cells and macrophages. Although there are two endogenous
ligands for PD1-PD-L1 and PD-L2, anti-tumor therapies have focused
on anti-PD-L1 antibodies. The complex of PD1 and PD-L1 inhibits
proliferation of CD8+ T cells and reduces the immune response
(Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al.,
2012, N Eng J Med 366:2455-65). Anti-PD-L1 antibodies have been
used for treatment of non-small cell lung cancer, melanoma,
colorectal cancer, renal-cell cancer, pancreatic cancer, gastric
cancer, ovarian cancer, breast cancer, and hematologic malignancies
(Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013, Clin
Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res
19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85;
Berger et al., 2008, Clin Cancer Res 14:13044-51).
Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX),
MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559
(BRISTOL-MYERS SQUIBB). Anti-PD-L1 antibodies are also commercially
available, for example from AFFYMETRIX EBIOSCIENCE (MIH1).
Cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152) is
also a member of the immunoglobulin superfamily that is expressed
exclusively on T-cells. CTLA4 acts to inhibit T-cell activation and
is reported to inhibit helper T-cell activity and enhance
regulatory T-cell immunosuppressive activity (Pardoll, 2012, Nature
Reviews Cancer 12:252-264). Although the precise mechanism of
action of CTL4-A remains under investigation, it has been suggested
that it inhibits T cell activation by outcompeting CD28 in binding
to CD80 and CD86, as well as actively delivering inhibitor signals
to the T cell (Pardoll, 2012, Nature Reviews Cancer 12:252-264).
Anti-CTL4A antibodies have been used in clinical trials for
treatment of melanoma, prostate cancer, small cell lung cancer,
non-small cell lung cancer (Robert & Ghiringhelli, 2009,
Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300;
Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med
11:89). A significant feature of anti-CTL4A is the kinetics of
anti-tumor effect, with a lag period of up to 6 months after
initial treatment required for physiologic response (Pardoll, 2012,
Nature Reviews Cancer 12:252-264). In some cases, tumors may
actually increase in size after treatment initiation, before a
reduction is seen (Pardoll, 2012, Nature Reviews Cancer
12:252-264).
Exemplary anti-CTLA4 antibodies include ipilimumab (Bristol-Myers
Squibb) and tremelimumab (PFIZER). Anti-PD1 antibodies are
commercially available, for example from ABCAM.RTM. (AB134090),
SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMO
SCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205,
MA1-35914). Ipilimumab has recently received FDA approval for
treatment of metastatic melanoma (Wada et al., 2013, J Transl Med
11:89).
The person of ordinary skill will realize that methods of
determining optimal dosages of checkpoint inhibitor antibodies to
administer to a patient in need thereof, either alone or in
combination with one or more other agents, may be determined by
standard dose-response and toxicity studies that are well known in
the art. In an exemplary embodiment, a checkpoint inhibitor
antibody may preferably be administered at about 0.3-10 mg/kg, or
the maximum tolerated dose, administered about every three weeks or
about every six weeks. Alternatively, the checkpoint inhibitor
antibody may be administered by an escalating dosage regimen
including administering a first dosage at about 3 mg/kg, a second
dosage at about 5 mg/kg, and a third dosage at about 9 mg/kg.
Alternatively, the escalating dosage regimen includes administering
a first dosage of checkpoint inhibitor antibody at about 5 mg/kg
and a second dosage at about 9 mg/kg. Another stepwise escalating
dosage regimen may include administering a first dosage of
checkpoint inhibitor antibody about 3 mg/kg, a second dosage of
about 3 mg/kg, a third dosage of about 5 mg/kg, a fourth dosage of
about 5 mg/kg, and a fifth dosage of about 9 mg/kg. In another
aspect, a stepwise escalating dosage regimen may include
administering a first dosage of 5 mg/kg, a second dosage of 5
mg/kg, and a third dosage of 9 mg/kg. Exemplary reported dosages of
checkpoint inhibitor mAbs include 3 mg/kg ipilimumab administered
every three weeks for four doses; 10 mg/kg ipilimumab every three
weeks for eight cycles; 10 mg/kg every three weeks for four cycles
then every 12 weeks for a total of three years; 10 mg/kg MK-3475
every two or every three weeks; 2 mg/kg MK-3475 every three weeks;
15 mg/kg tremilimumab every three months; 0.1, 0.3, 1, 3 or 10
mg/kg nivolumab every two weeks for up to 96 weeks; 0.3, 1, 3, or
10 mg/kg BMS-936559 every two weeks for up to 96 weeks (Kyi &
Postow, Oct. 23, 2013, FEBS Lett [Epub ahead of print]; Callahan
& Wolchok, 2013, J Leukoc Biol 94:41-53).
These and other known agents that stimulate immune response to
tumors and/or pathogens may be used in combination with leukocyte
redirecting bispecific antibodies alone or in further combination
with an interferon, such as interferon-.alpha., and/or an
antibody-drug conjugate for improved cancer therapy. Other known
co-stimulatory pathway modulators that may be used in combination
include, but are not limited to, agatolimod, belatacept,
blinatumomab, CD40 ligand, anti-B7-1 antibody, anti-B7-2 antibody,
anti-B7-H4 antibody, AG4263, eritoran, anti-OX40 antibody, ISF-154,
and SGN-70; B7-1, B7-2, ICAM-1, ICAM-2, ICAM-3, CD48, LFA-3, CD30
ligand, CD40 ligand, heat stable antigen, B7h, OX40 ligand, LIGHT,
CD70 and CD24.
In certain embodiments, anti-KIR antibodies may also be used in
combination with leukocyte-redirecting bsAbs, interferons, ADCs
and/or checkpoint inhibitor antibodies. NK cells mediate anti-tumor
and anti-infectious agent activity by spontaneous cytotoxicity and
by ADCC when activated by antibodies (Kohrt et al., 2013, Blood,
[Epub ahead of print Dec. 10, 2013]). The degree of cytotoxic
response is determined by a balance of inhibitory and activating
signals received by the NK cells (Kohrt et al., 2013). The killer
cell immunoglobulin-like receptor (KIR) mediates an inhibitory
signal that decreases NK cell response. Anti-KIR antibodies, such
as lirilumab (Innate Pharma) and IPH2101 (Innate Pharma) have
demonstrated anti-tumor activity in multiple myeloma (Benson et
al., 2012, Blood 120:4324-33). In vitro, anti-KIR antibodies
prevent the tolerogenic interaction of NK cells with target cells
and augments the NK cell cytotoxic response to tumor cells (Kohrt
et al., 2013). In vivo, in combination with rituximab (anti-CD20),
anti-KIR antibodies at a dose of 0.5 mg/kg induced enhanced NK
cell-mediated, rituximab-dependent cytotoxicity against lymphoma
tumors (Kohrt et al., 2013). Anti-KIR mAbs may be combined with
ADCs, leukocyte-redirecting bsAbs, interferons and/or checkpoint
inhibitor antibodies to potentiate cytotoxicity to tumor cells or
pathogenic organisms.
General Antibody Techniques
Techniques for preparing monoclonal antibodies against virtually
any target antigen are well known in the art. See, for example,
Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al.
(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991). Briefly, monoclonal antibodies can be
obtained by injecting mice with a composition comprising an
antigen, removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
MAbs can be isolated and purified from hybridoma cultures by a
variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10,
pages 79-104 (The Humana Press, Inc. 1992).
After the initial raising of antibodies to the immunogen, the
antibodies can be sequenced and subsequently prepared by
recombinant techniques. Humanization and chimerization of murine
antibodies and antibody fragments are well known to those skilled
in the art. The use of antibody components derived from humanized,
chimeric or human antibodies obviates potential problems associated
with the immunogenicity of murine constant regions.
Chimeric Antibodies
A chimeric antibody is a recombinant protein in which the variable
regions of a human antibody have been replaced by the variable
regions of, for example, a mouse antibody, including the
complementarity-determining regions (CDRs) of the mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased
stability when administered to a subject. General techniques for
cloning murine immunoglobulin variable domains are disclosed, for
example, in Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833
(1989). Techniques for constructing chimeric antibodies are well
known to those of skill in the art. As an example, Leung et al.,
Hybridoma 13:469 (1994), produced an LL2 chimera by combining DNA
sequences encoding the V.sub..kappa. and V.sub.H domains of murine
LL2, an anti-CD22 monoclonal antibody, with respective human
.kappa. and IgG.sub.1 constant region domains.
Humanized Antibodies
Techniques for producing humanized MAbs are well known in the art
(see, e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al.,
Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988),
Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu,
Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun.
150: 2844 (1993)). A chimeric or murine monoclonal antibody may be
humanized by transferring the mouse CDRs from the heavy and light
variable chains of the mouse immunoglobulin into the corresponding
variable domains of a human antibody. The mouse framework regions
(FR) in the chimeric monoclonal antibody are also replaced with
human FR sequences. As simply transferring mouse CDRs into human
FRs often results in a reduction or even loss of antibody affinity,
additional modification might be required in order to restore the
original affinity of the murine antibody. This can be accomplished
by the replacement of one or more human residues in the FR regions
with their murine counterparts to obtain an antibody that possesses
good binding affinity to its epitope. See, for example, Tempest et
al., Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239:
1534 (1988). Generally, those human FR amino acid residues that
differ from their murine counterparts and are located close to or
touching one or more CDR amino acid residues would be candidates
for substitution.
Human Antibodies
Methods for producing fully human antibodies using either
combinatorial approaches or transgenic animals transformed with
human immunoglobulin loci are known in the art (e.g., Mancini et
al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005,
Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset,
2003, Curr. Opin. Phamacol. 3:544-50). A fully human antibody also
can be constructed by genetic or chromosomal transfection methods,
as well as phage display technology, all of which are known in the
art. See for example, McCafferty et al., Nature 348:552-553 (1990).
Such fully human antibodies are expected to exhibit even fewer side
effects than chimeric or humanized antibodies and to function in
vivo as essentially endogenous human antibodies. In certain
embodiments, the claimed methods and procedures may utilize human
antibodies produced by such techniques.
In one alternative, the phage display technique may be used to
generate human antibodies (e.g., Dantas-Barbosa et al., 2005,
Genet. Mol. Res. 4:126-40). Human antibodies may be generated from
normal humans or from humans that exhibit a particular disease
state, such as cancer (Dantas-Barbosa et al., 2005). The advantage
to constructing human antibodies from a diseased individual is that
the circulating antibody repertoire may be biased towards
antibodies against disease-associated antigens.
In one non-limiting example of this methodology, Dantas-Barbosa et
al. (2005) constructed a phage display library of human Fab
antibody fragments from osteosarcoma patients. Generally, total RNA
was obtained from circulating blood lymphocytes (Id.). Recombinant
Fab were cloned from the .mu., .gamma. and .kappa. chain antibody
repertoires and inserted into a phage display library (Id.). RNAs
were converted to cDNAs and used to make Fab cDNA libraries using
specific primers against the heavy and light chain immunoglobulin
sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97). Library
construction was performed according to Andris-Widhopf et al.
(2000, In: PHAGE DISPLAY LABORATORY MANUAL, Barbas et al. (eds),
1.sup.st edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. pp. 9.1 to 9.22). The final Fab fragments were
digested with restriction endonucleases and inserted into the
bacteriophage genome to make the phage display library. Such
libraries may be screened by standard phage display methods, as
known in the art (see, e.g., Pasqualini and Ruoslahti, 1996, Nature
380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med.
43:159-162).
Phage display can be performed in a variety of formats, for their
review, see e.g. Johnson and Chiswell, Current Opinion in
Structural Biology 3:5564-571 (1993). Human antibodies may also be
generated by in vitro activated B cells. See U.S. Pat. Nos.
5,567,610 and 5,229,275, incorporated herein by reference in their
entirety. The skilled artisan will realize that these techniques
are exemplary and any known method for making and screening human
antibodies or antibody fragments may be utilized.
In another alternative, transgenic animals that have been
genetically engineered to produce human antibodies may be used to
generate antibodies against essentially any immunogenic target,
using standard immunization protocols. Methods for obtaining human
antibodies from transgenic mice are disclosed by Green et al.,
Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994),
and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example
of such a system is the XENOMOUSE.RTM. (e.g., Green et al., 1999,
J. Immunol. Methods 231:11-23) from Abgenix (Fremont, Calif.). In
the XENOMOUSE.RTM. and similar animals, the mouse antibody genes
have been inactivated and replaced by functional human antibody
genes, while the remainder of the mouse immune system remains
intact.
The XENOMOUSE.RTM. was transformed with germline-configured YACs
(yeast artificial chromosomes) that contained portions of the human
IgH and Igkappa loci, including the majority of the variable region
sequences, along accessory genes and regulatory sequences. The
human variable region repertoire may be used to generate antibody
producing B cells, which may be processed into hybridomas by known
techniques. A XENOMOUSE.RTM. immunized with a target antigen will
produce human antibodies by the normal immune response, which may
be harvested and/or produced by standard techniques discussed
above. A variety of strains of XENOMOUSE.RTM. are available, each
of which is capable of producing a different class of antibody.
Transgenically produced human antibodies have been shown to have
therapeutic potential, while retaining the pharmacokinetic
properties of normal human antibodies (Green et al., 1999). The
skilled artisan will realize that the claimed compositions and
methods are not limited to use of the XENOMOUSE.RTM. system but may
utilize any transgenic animal that has been genetically engineered
to produce human antibodies.
Antibody Cloning and Production
Various techniques, such as production of chimeric or humanized
antibodies, may involve procedures of antibody cloning and
construction. The antigen-binding V.kappa. (variable light chain)
and V.sub.H (variable heavy chain) sequences for an antibody of
interest may be obtained by a variety of molecular cloning
procedures, such as RT-PCR, 5'-RACE, and cDNA library screening.
The V genes of an antibody from a cell that expresses a murine
antibody can be cloned by PCR amplification and sequenced. To
confirm their authenticity, the cloned V.sub.L and V.sub.H genes
can be expressed in cell culture as a chimeric Ab as described by
Orlandi et al., (Proc. Natl. Acad. Sci. USA, 86: 3833 (1989)).
Based on the V gene sequences, a humanized antibody can then be
designed and constructed as described by Leung et al. (Mol.
Immunol., 32: 1413 (1995)).
cDNA can be prepared from any known hybridoma line or transfected
cell line producing a murine antibody by general molecular cloning
techniques (Sambrook et al., Molecular Cloning, A laboratory
manual, 2.sup.nd Ed (1989)). The V.kappa. sequence for the antibody
may be amplified using the primers VK1BACK and VK1FOR (Orlandi et
al., 1989) or the extended primer set described by Leung et al.
(BioTechniques, 15: 286 (1993)). The V.sub.H sequences can be
amplified using the primer pair VH1BACK/VH1FOR (Orlandi et al.,
1989) or the primers annealing to the constant region of murine IgG
described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized V
genes can be constructed by a combination of long oligonucleotide
template syntheses and PCR amplification as described by Leung et
al. (Mol. Immunol., 32: 1413 (1995)).
PCR products for V.kappa. can be subcloned into a staging vector,
such as a pBR327-based staging vector, VKpBR, that contains an Ig
promoter, a signal peptide sequence and convenient restriction
sites. PCR products for V.sub.H can be subcloned into a similar
staging vector, such as the pBluescript-based VHpBS. Expression
cassettes containing the V.kappa. and V.sub.H sequences together
with the promoter and signal peptide sequences can be excised from
VKpBR and VHpBS and ligated into appropriate expression vectors,
such as pKh and pG1g, respectively (Leung et al., Hybridoma, 13:469
(1994)). The expression vectors can be co-transfected into an
appropriate cell and supernatant fluids monitored for production of
a chimeric, humanized or human antibody. Alternatively, the
V.sub..kappa. and V.sub.H expression cassettes can be excised and
subcloned into a single expression vector, such as pdHL2, as
described by Gillies et al. (J. Immunol. Methods 125:191 (1989) and
also shown in Losman et al., Cancer, 80:2660 (1997)).
In an alternative embodiment, expression vectors may be transfected
into host cells that have been pre-adapted for transfection, growth
and expression in serum-free medium. Exemplary cell lines that may
be used include the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see,
e.g., U.S. Pat. Nos. 7,531,327; 7,537,930 and 7,608,425; the
Examples section of each of which is incorporated herein by
reference). These exemplary cell lines are based on the Sp2/0
myeloma cell line, transfected with a mutant Bcl-EEE gene, exposed
to methotrexate to amplify transfected gene sequences and
pre-adapted to serum-free cell line for protein expression.
Antibody Fragments
Antibody fragments which recognize specific epitopes can be
generated by known techniques. Antibody fragments are antigen
binding portions of an antibody, such as F(ab').sub.2, Fab',
F(ab).sub.2, Fab, Fv, scFv and the like. F(ab').sub.2 fragments can
be produced by pepsin digestion of the antibody molecule and Fab'
fragments can be generated by reducing disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab' expression libraries
can be constructed (Huse et al., 1989, Science, 246:1274-1281) to
allow rapid and easy identification of monoclonal Fab' fragments
with the desired specificity. F(ab).sub.2 fragments may be
generated by papain digestion of an antibody.
A single chain Fv molecule (scFv) comprises a VL domain and a VH
domain. The VL and VH domains associate to form a target binding
site. These two domains are further covalently linked by a peptide
linker (L). Methods for making scFv molecules and designing
suitable peptide linkers are described in U.S. Pat. No. 4,704,692;
U.S. Pat. No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and
Bird and Walker, TIBTECH, 9: 132-137 (1991).
Techniques for producing single domain antibodies (DABs or VHH) are
also known in the art, as disclosed for example in Cossins et al.
(2006, Prot Express Purif 51:253-259), incorporated herein by
reference. Single domain antibodies may be obtained, for example,
from camels, alpacas or llamas by standard immunization techniques.
(See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al.,
J Immunol Methods 281:161-75, 2003; Maass et al., J Immunol Methods
324:13-25, 2007). The VHH may have potent antigen-binding capacity
and can interact with novel epitopes that are inacessible to
conventional VH-VL pairs. (Muyldermans et al., 2001). Alpaca serum
IgG contains about 50% camelid heavy chain only IgG antibodies
(HCAbs) (Maass et al., 2007). Alpacas may be immunized with known
antigens, such as TNF-.alpha., and VHHs can be isolated that bind
to and neutralize the target antigen (Maass et al., 2007). PCR
primers that amplify virtually all alpaca VHH coding sequences have
been identified and may be used to construct alpaca VHH phage
display libraries, which can be used for antibody fragment
isolation by standard biopanning techniques well known in the art
(Maass et al., 2007). In certain embodiments, anti-pancreatic
cancer VHH antibody fragments may be utilized in the claimed
compositions and methods.
An antibody fragment can be prepared by proteolytic hydrolysis of
the full length antibody or by expression in E. coli or another
host of the DNA coding for the fragment. An antibody fragment can
be obtained by pepsin or papain digestion of full length antibodies
by conventional methods. These methods are described, for example,
by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and
references contained therein. Also, see Nisonoff et al., Arch
Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119
(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422
(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and
2.10.-2.10.4.
Antibody Allotypes
Immunogenicity of therapeutic antibodies is associated with
increased risk of infusion reactions and decreased duration of
therapeutic response (Baert et al., 2003, N Engl J Med 348:602-08).
The extent to which therapeutic antibodies induce an immune
response in the host may be determined in part by the allotype of
the antibody (Stickler et al., 2011, Genes and Immunity 12:213-21).
Antibody allotype is related to amino acid sequence variations at
specific locations in the constant region sequences of the
antibody. The allotypes of IgG antibodies containing a heavy chain
.gamma.-type constant region are designated as Gm allotypes (1976,
J Immunol 117:1056-59).
For the common IgG1 human antibodies, the most prevalent allotype
is G1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21).
However, the G1m3 allotype also occurs frequently in Caucasians
(Stickler et al., 2011). It has been reported that G1m1 antibodies
contain allotypic sequences that tend to induce an immune response
when administered to non-G1m1 (nG1m1) recipients, such as G1m3
patients (Stickler et al., 2011). Non-G1m1 allotype antibodies are
not as immunogenic when administered to G1m1 patients (Stickler et
al., 2011).
The human G1m1 allotype comprises the amino acids aspartic acid at
Kabat position 356 and leucine at Kabat position 358 in the CH3
sequence of the heavy chain IgG1. The nG1m1 allotype comprises the
amino acids glutamic acid at Kabat position 356 and methionine at
Kabat position 358. Both G1m1 and nG1m1 allotypes comprise a
glutamic acid residue at Kabat position 357 and the allotypes are
sometimes referred to as DEL and EEM allotypes. A non-limiting
example of the heavy chain constant region sequences for G1m1 and
nG1m1 allotype antibodies is shown for the exemplary antibodies
rituximab (SEQ ID NO:85) and veltuzumab (SEQ ID NO:86).
TABLE-US-00001 Rituximab heavy chain variable region sequence (SEQ
ID NO: 85) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable
region (SEQ ID NO: 86)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence
variations characteristic of IgG allotypes and their effect on
immunogenicity. They reported that the G1m3 allotype is
characterized by an arginine residue at Kabat position 214,
compared to a lysine residue at Kabat 214 in the G1m17 allotype.
The nG1m1,2 allotype was characterized by glutamic acid at Kabat
position 356, methionine at Kabat position 358 and alanine at Kabat
position 431. The G1m1,2 allotype was characterized by aspartic
acid at Kabat position 356, leucine at Kabat position 358 and
glycine at Kabat position 431. In addition to heavy chain constant
region sequence variants, Jefferis and Lefranc (2009) reported
allotypic variants in the kappa light chain constant region, with
the Km1 allotype characterized by valine at Kabat position 153 and
leucine at Kabat position 191, the Km1,2 allotype by alanine at
Kabat position 153 and leucine at Kabat position 191, and the Km3
allotype characterized by alanine at Kabat position 153 and valine
at Kabat position 191.
With regard to therapeutic antibodies, veltuzumab and rituximab
are, respectively, humanized and chimeric IgG1 antibodies against
CD20, of use for therapy of a wide variety of hematological
malignancies and/or autoimmune diseases. Table 1 compares the
allotype sequences of rituximab vs. veltuzumab. As shown in Table
1, rituximab (G1m17,1) is a DEL allotype IgG1, with an additional
sequence variation at Kabat position 214 (heavy chain CH1) of
lysine in rituximab vs. arginine in veltuzumab. It has been
reported that veltuzumab is less immunogenic in subjects than
rituximab (see, e.g., Morchhauser et al., 2009, J Clin Oncol
27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &
Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed
to the difference between humanized and chimeric antibodies.
However, the difference in allotypes between the EEM and DEL
allotypes likely also accounts for the lower immunogenicity of
veltuzumab.
TABLE-US-00002 TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy
chain position and associated allotypes Complete 356/358 allotype
214 (allotype) (allotype) 431 (allotype) Rituximab G1m17,1 K 17 D/L
1 A -- Veltuzumab G1m3 R 3 E/M -- A --
In order to reduce the immunogenicity of therapeutic antibodies in
individuals of nG1m1 genotype, it is desirable to select the
allotype of the antibody to correspond to the G1m3 allotype,
characterized by arginine at Kabat 214, and the nG1m1,2
null-allotype, characterized by glutamic acid at Kabat position
356, methionine at Kabat position 358 and alanine at Kabat position
431. Surprisingly, it was found that repeated subcutaneous
administration of G1m3 antibodies over a long period of time did
not result in a significant immune response. In alternative
embodiments, the human IgG4 heavy chain in common with the G1m3
allotype has arginine at Kabat 214, glutamic acid at Kabat 356,
methionine at Kabat 359 and alanine at Kabat 431. Since
immunogenicity appears to relate at least in part to the residues
at those locations, use of the human IgG4 heavy chain constant
region sequence for therapeutic antibodies is also a preferred
embodiment. Combinations of G1m3 IgG1 antibodies with IgG4
antibodies may also be of use for therapeutic administration.
Known Antibodies
Target Antigens and Exemplary Antibodies
In a preferred embodiment, antibodies are used that recognize
and/or bind to antigens that are expressed at high levels on target
cells and that are expressed predominantly or exclusively on
diseased cells versus normal tissues. Exemplary antibodies of use
for therapy of, for example, cancer include but are not limited to
LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20,
anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20),
lambrolizumab (anti-PD1), nivolumab (anti-PD1), MK-3475 (anti-PD1),
AMP-224 (anti-PD1), pidilizumab (anti-PD1), MDX-1105 (anti-PD-L1),
MEDI4736 (anti-PD-L1), MPDL3280A (anti-PD-L1), BMS-936559
(anti-PD-L1), ipilimumab (anti-CTLA4), trevilizumab (anti-CTL4A),
RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)),
PAM4 or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen
(CEA, also known as CD66e or CEACAM5), MN-15 or MN-3
(anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (an
anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72
(e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specific
membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B
(anti-PSMA), G250 (an anti-carbonic anhydrase IX MAb), L243
(anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),
cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan
(anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4
(aka clivatuzumab, anti-mucin), BWA-3 (anti-histone H2A/H4), LG2-1
(anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone
H2B), LG11-2 (anti-histone H2B), LG2-2 (anti-histone H2B), and
trastuzumab (anti-ErbB2). Such antibodies are known in the art
(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;
6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;
7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;
7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S.
Patent Application Publ. No. 20050271671; 20060193865; 20060210475;
20070087001; the Examples section of each incorporated herein by
reference.) Specific known antibodies of use include hPAM4 (U.S.
Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S.
Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1 (U.S.
Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.
Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S.
Pat. No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S.
patent application Ser. No. 12/772,645), hRS7 (U.S. Pat. No.
7,238,785), hMN-3 (U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S.
patent application Ser. No. 11/983,372, deposited as ATCC PTA-4405
and PTA-4406) and D2/B (WO 2009/130575) the text of each recited
patent or application is incorporated herein by reference with
respect to the Figures and Examples sections.
Other useful antigens that may be targeted using the described
conjugates include carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp,
HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,
CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21,
CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L,
CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74,
CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154,
CEACAM5, CEACAM6, CTLA4, alpha-fetoprotein (AFP), VEGF (e.g.,
AVASTIN.RTM., fibronectin splice variant), ED-B fibronectin (e.g.,
L19), EGP-1 (TROP-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1)
(e.g., ERBITUX.RTM.), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate
receptor, Ga 733, GRO-.beta., HMGB-1, hypoxia inducible factor
(HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),
IFN-.gamma., IFN-.alpha., IFN-.beta., IFN-.lamda., IL-2R, IL-4R,
IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12,
IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24,
gangliosides, HCG, the HLA-DR antigen to which L243 binds, CD66
antigens, i.e., CD66a-d or a combination thereof, MAGE, mCRP,
MCP-1, MIP-1A, MIP-1B, macrophage migration-inhibitory factor
(MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor
(PlGF), PSA (prostate-specific antigen), PSMA, PAM4 antigen, PD1
receptor, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin,
S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens,
tumor necrosis antigens, tumor angiogenesis antigens, TNF-.alpha.,
TRAIL receptor (R1 and R2), TROP-2, VEGFR, RANTES, T101, as well as
cancer stem cell antigens, complement factors C3, C3a, C3b, C5a,
C5, and an oncogene product.
A comprehensive analysis of suitable antigen (Cluster Designation,
or CD) targets on hematopoietic malignant cells, as shown by flow
cytometry and which can be a guide to selecting suitable antibodies
for immunotherapy, is Craig and Foon, Blood prepublished online
Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.
The CD66 antigens consist of five different glycoproteins with
similar structures, CD66a-e, encoded by the carcinoembryonic
antigen (CEA) gene family members, BCG, CGM6, NCA, CGM1 and CEA,
respectively. These CD66 antigens (e.g., CEACAM6) are expressed
mainly in granulocytes, normal epithelial cells of the digestive
tract and tumor cells of various tissues. Also included as suitable
targets for cancers are cancer testis antigens, such as NY-ESO-1
(Theurillat et al., Int. J. Cancer 2007; 120(11):2411-7), as well
as CD79a in myeloid leukemia (Kozlov et al., Cancer Genet.
Cytogenet. 2005; 163(1):62-7) and also B-cell diseases, and CD79b
for non-Hodgkin's lymphoma (Poison et al., Blood 110(2):616-623). A
number of the aforementioned antigens are disclosed in U.S.
Provisional Application Ser. No. 60/426,379, entitled "Use of
Multi-specific, Non-covalent Complexes for Targeted Delivery of
Therapeutics," filed Nov. 15, 2002. Cancer stem cells, which are
ascribed to be more therapy-resistant precursor malignant cell
populations (Hill and Perris, J. Natl. Cancer Inst. 2007;
99:1435-40), have antigens that can be targeted in certain cancer
types, such as CD133 in prostate cancer (Maitland et al., Ernst
Schering Found. Sympos. Proc. 2006; 5:155-79), non-small-cell lung
cancer (Donnenberg et al., J. Control Release 2007; 122(3):385-91),
and glioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5),
and CD44 in colorectal cancer (Dalerba er al., Proc. Natl. Acad.
Sci. USA 2007; 104(24)10158-63), pancreatic cancer (Li et al.,
Cancer Res. 2007; 67(3):1030-7), and in head and neck squamous cell
carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;
104(3)973-8).
Anti-cancer antibodies have been demonstrated to bind to histones
in some case. Kato et al. (1991, Hum Antibodies Hybridomas
2:94-101) reported that the lung cancer-specific human monoclonal
antibody HB4C5 binds to histone H2B. Garzelli et al. (1994, Immunol
Lett 39:277-82) observed that Epstein-Barr virus-transformed human
B lymphocytes produce natural antibodies to histones. In certain
embodiments, antibodies against histones may be of use in the
subject combinations. Known anti-histone antibodies include, but
are not limited to, BWA-3 (anti-histone H2A/H4), LG2-1
(anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone
H2B), LG11-2 (anti-histone H2B), and LG2-2 (anti-histone H2B) (see,
e.g., Monestier et al., 1991, Eur J Immunol 21:1725-31; Monestier
et al., 1993, Molec Immunol 30:1069-75).
For multiple myeloma therapy, suitable targeting antibodies have
been described against, for example, CD38 and CD138 (Stevenson, Mol
Med 2006; 12(11-12):345-346; Tassone et al., Blood 2004;
104(12):3688-96), CD74 (Stein et al., ibid.), CS1 (Tai et al.,
Blood 2008; 112(4):1329-37, and CD40 (Tai et al., 2005; Cancer Res.
65(13):5898-5906).
Macrophage migration inhibitory factor (MIF) is an important
regulator of innate and adaptive immunity and apoptosis. It has
been reported that CD74 is the endogenous receptor for MIF (Leng et
al., 2003, J Exp Med 197:1467-76). The therapeutic effect of
antagonistic anti-CD74 antibodies on MIF-mediated intracellular
pathways may be of use for treatment of a broad range of disease
states, such as cancers of the bladder, prostate, breast, lung,
colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al.,
2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma
52:1446-54). Milatuzumab (hLL1) is an exemplary anti-CD74 antibody
of therapeutic use for treatment of MIF-mediated diseases.
An example of a most-preferred antibody/antigen pair is LL1, an
anti-CD74 MAb (invariant chain, class II-specific chaperone, Ii)
(see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; the Examples
section of each incorporated herein by reference). The CD74 antigen
is highly expressed on B-cell lymphomas (including multiple
myeloma) and leukemias, certain T-cell lymphomas, melanomas,
colonic, lung, and renal cancers, glioblastomas, and certain other
cancers (Ong et al., Immunology 98:296-302 (1999)). A review of the
use of CD74 antibodies in cancer is contained in Stein et al., Clin
Cancer Res. 2007 Sep. 15; 13(18 Pt 2):5556s-5563s, incorporated
herein by reference. The diseases that are preferably treated with
anti-CD74 antibodies include, but are not limited to, non-Hodgkin's
lymphoma, Hodgkin's disease, melanoma, lung, renal, colonic
cancers, glioblastome multiforme, histiocytomas, myeloid leukemias,
and multiple myeloma.
In various embodiments, the claimed methods and compositions may
utilize any of a variety of antibodies known in the art. Antibodies
of use may be commercially obtained from a number of known sources.
For example, a variety of antibody secreting hybridoma lines are
available from the American Type Culture Collection (ATCC,
Manassas, Va.). A large number of antibodies against various
disease targets, including but not limited to tumor-associated
antigens, have been deposited at the ATCC and/or have published
variable region sequences and are available for use in the claimed
methods and compositions. See, e.g., U.S. Pat. Nos. 7,312,318;
7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060;
7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498;
7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241;
6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107;
6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645;
6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681;
6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405;
6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511;
6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780;
6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711;
6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307;
6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908; 6,689,607;
6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344;
6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294;
6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572,856;
6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;
6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;
6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;
6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206'
6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;
6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;
6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444;
6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459;
6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914;
6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;
5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136;
5,587,459; 5,443,953, 5,525,338, the Examples section of each of
which is incorporated herein by reference. These are exemplary only
and a wide variety of other antibodies and their hybridomas are
known in the art. The skilled artisan will realize that antibody
sequences or antibody-secreting hybridomas against almost any
disease-associated antigen may be obtained by a simple search of
the ATCC, NCBI and/or USPTO databases for antibodies against a
selected disease-associated target of interest. The antigen binding
domains of the cloned antibodies may be amplified, excised, ligated
into an expression vector, transfected into an adapted host cell
and used for protein production, using standard techniques well
known in the art (see, e.g., U.S. Pat. Nos. 7,531,327; 7,537,930;
7,608,425 and 7,785,880, the Examples section of each of which is
incorporated herein by reference).
In other embodiments, the antibody complexes bind to a MHC class I,
MHC class II or accessory molecule, such as CD40, CD54, CD80 or
CD86. The antibody complex also may bind to a leukocyte activation
cytokine, or to a cytokine mediator, such as NF-.kappa.B.
In certain embodiments, one of the two different targets may be a
cancer cell receptor or cancer-associated antigen, particularly one
that is selected from the group consisting of B-cell lineage
antigens (CD19, CD20, CD21, CD22, CD23, etc.), VEGF, VEGFR, EGFR,
carcinoembryonic antigen (CEA), placental growth factor (PlGF),
tenascin, HER-2/neu, EGP-1, EGP-2, CD25, CD30, CD33, CD38, CD40,
CD45, CD52, CD74, CD80, CD138, NCA66, CEACAM1, CEACAM6
(carcinoembryonic antigen-related cellular adhesion molecule 6),
MUC1, MUC2, MUC3, MUC4, MUC16, IL-6, .alpha.-fetoprotein (AFP), A3,
CA125, colon-specific antigen-p (CSAp), folate receptor, HLA-DR,
human chorionic gonadotropin (HCG), Ia, EL-2, insulin-like growth
factor (IGF) and IGF receptor, KS-1, Le(y), MAGE, necrosis
antigens, PAM-4, prostatic acid phosphatase (PAP), Pr1, prostate
specific antigen (PSA), prostate specific membrane antigen (PSMA),
5100, T101, TAC, TAG72, TRAIL receptors, and carbonic anhydrase
IX.
Immunoconjugates
In certain embodiments, the antibodies or fragments thereof may be
conjugated to one or more therapeutic or diagnostic agents. The
therapeutic agents do not need to be the same but can be different,
e.g. a drug and a radioisotope. For example, .sup.131I can be
incorporated into a tyrosine of an antibody or fusion protein and a
drug attached to an epsilon amino group of a lysine residue.
Therapeutic and diagnostic agents also can be attached, for example
to reduced SH groups and/or to carbohydrate side chains. Many
methods for making covalent or non-covalent conjugates of
therapeutic or diagnostic agents with antibodies or fusion proteins
are known in the art and any such known method may be utilized.
A therapeutic or diagnostic agent can be attached at the hinge
region of a reduced antibody component via disulfide bond
formation. Alternatively, such agents can be attached using a
heterobifunctional cross-linker, such as N-succinyl
3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56:
244 (1994). General techniques for such conjugation are well-known
in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages
187-230 (Wiley-Liss, Inc. 1995); Price, "Production and
Characterization of Synthetic Peptide-Derived Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995). Alternatively, the therapeutic or
diagnostic agent can be conjugated via a carbohydrate moiety in the
Fc region of the antibody. The carbohydrate group can be used to
increase the loading of the same agent that is bound to a thiol
group, or the carbohydrate moiety can be used to bind a different
therapeutic or diagnostic agent.
Methods for conjugating peptides to antibody components via an
antibody carbohydrate moiety are well-known to those of skill in
the art. See, for example, Shih et al., Int. J. Cancer 41: 832
(1988); Shih et al., Int. J. Cancer 46: 1101 (1990); and Shih et
al., U.S. Pat. No. 5,057,313, incorporated herein in their entirety
by reference. The general method involves reacting an antibody
component having an oxidized carbohydrate portion with a carrier
polymer that has at least one free amine function. This reaction
results in an initial Schiff base (imine) linkage, which can be
stabilized by reduction to a secondary amine to form the final
conjugate.
The Fc region may be absent if the antibody used as the antibody
component of the immunoconjugate is an antibody fragment. However,
it is possible to introduce a carbohydrate moiety into the light
chain variable region of a full length antibody or antibody
fragment. See, for example, Leung et al., J. Immunol. 154: 5919
(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et
al., U.S. Pat. No. 6,254,868, incorporated herein by reference in
their entirety. The engineered carbohydrate moiety is used to
attach the therapeutic or diagnostic agent.
In some embodiments, a chelating agent may be attached to an
antibody, antibody fragment or fusion protein and used to chelate a
therapeutic or diagnostic agent, such as a radionuclide. Exemplary
chelators include but are not limited to DTPA (such as Mx-DTPA),
DOTA, TETA, NETA or NOTA. Methods of conjugation and use of
chelating agents to attach metals or other ligands to proteins are
well known in the art (see, e.g., U.S. Pat. No. 7,563,433, the
Examples section of which is incorporated herein by reference).
In certain embodiments, radioactive metals or paramagnetic ions may
be attached to proteins or peptides by reaction with a reagent
having a long tail, to which may be attached a multiplicity of
chelating groups for binding ions. Such a tail can be a polymer
such as a polylysine, polysaccharide, or other derivatized or
derivatizable chains having pendant groups to which can be bound
chelating groups such as, e.g., ethylenediaminetetraacetic acid
(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,
polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and
like groups known to be useful for this purpose.
Chelates may be directly linked to antibodies or peptides, for
example as disclosed in U.S. Pat. No. 4,824,659, incorporated
herein in its entirety by reference. Particularly useful
metal-chelate combinations include 2-benzyl-DTPA and its monomethyl
and cyclohexyl analogs, used with diagnostic isotopes in the
general energy range of 60 to 4,000 keV, such as .sup.125I,
.sup.131I, .sup.123I, .sup.124I, .sup.62Cu, .sup.64Cu, .sup.18F,
.sup.111In, .sup.67Ga, .sup.68Ga, .sup.99mTc, .sup.94mTc, .sup.11C,
.sup.13N, .sup.15O, .sup.76Br, for radioimaging. The same chelates,
when complexed with non-radioactive metals, such as manganese, iron
and gadolinium are useful for MM. Macrocyclic chelates such as
NOTA, DOTA, and TETA are of use with a variety of metals and
radiometals, most particularly with radionuclides of gallium,
yttrium and copper, respectively. Such metal-chelate complexes can
be made very stable by tailoring the ring size to the metal of
interest. Other ring-type chelates such as macrocyclic polyethers,
which are of interest for stably binding nuclides, such as
.sup.223Ra for RAIT are encompassed.
More recently, methods of .sup.18F-labeling of use in PET scanning
techniques have been disclosed, for example by reaction of F-18
with a metal or other atom, such as aluminum. The .sup.18F-Al
conjugate may be complexed with chelating groups, such as DOTA,
NOTA or NETA that are attached directly to antibodies or used to
label targetable constructs in pre-targeting methods. Such F-18
labeling techniques are disclosed in U.S. Pat. No. 7,563,433, the
Examples section of which is incorporated herein by reference.
In specific preferred embodiments, an immunoconjugate may be an
antibody-drug conjugate (ADC). Two exemplary drugs of use in ADC
production are SN-38 and a prodrug form of 2-pyrrolinodoxorubicine
(P2PDox). Compositions and methods of production of
SN-38-conjugated ADCs are disclosed, for example, in U.S. Pat. Nos.
7,999,083; 8,080,250; 8,741,300; 8,759,496, the Figures and
Examples section of each of which are incorporated herein by
reference. Compositions and methods of production of P2PDox ADCs
are disclosed, for example, in U.S. Pat. No. 8,877,101, the Figures
and Examples section of which are incorporated herein by
reference.
Methods of Producing Bispecific Antibodies
In various embodiments, the subject combination therapy may utilize
one or more bispecific antibodies (bsAbs), such as a leukocyte
redirecting bsAb. A bispecific antibody as used herein is an
antibody that contains binding sites for two different antigens, or
two different epitopes on the same antigen. An antibody that can
only bind to a single epitope on a single antigen is monospecific,
regardless of the number of antigen-binding sites on the antibody
molecule.
Early attempts at bispecific antibody construction either utilized
chemical cross-linking or hybrid hybridomas or quadromas to join
the two halves of two different antibodies together (e.g., Staerz
et al., 1985, Nature 314:628-31; Milstein and Cuello, Nature 1983;
305:537-540; Karpovsky et al., 1984, J Exp Med 160:1686-701).
Although the techniques work to make bsAbs, various production
problems made use of such complexes difficult, such as the
production of mixed populations containing different combinations
of antigen-binding sites, difficulty in protein expression, the
need to purify the bsAb of interest, low yields, expense of
production, etc.
More recent approaches have utilized genetically engineered
constructs that are capable of producing homogeneous products of
single bsAbs, without the need for extensive purification to remove
unwanted byproducts. Such constructs have included tandem scFv,
diabodies, tandem diabodies, dual variable domain antibodies and
heterodimerization using a motif such as Ch1/Ck domain or DNL.TM.
(Chames & Baty, 2009, Curr Opin Drug Discov Devel 12:276-83;
Chames & Baty, mAbs 1:539-47).
Triomabs is a variation on the quadroma approach that use a
combination of mouse IgG2a and rat IgG2b antibodies to
preferentially produce the recombinant antibody, compared to the
random pairing typically seen in rat/rat or mouse/mouse quadromas
(Chames & Baty, mAbs 1:539-47). An anti-CD3.times.anti-EpCAM
bsAb (catumaxomab) created by this technique was able to
efficiently recruit macrophages and NK cells and to activate T
cells (Chames & Baty, mAbs 1:539-47). As discussed above,
catumaxomab has been approved in Europe for treatment of malignant
ascites in patients with EpCAM positive carcinomas (Chames &
Baty, mAbs 1:539-47). Surprisingly, the recombinant bsAb was
reported to induce only moderate anti-mouse and anti-rat responses
in humans (Chames & Baty, mAbs 1:539-47), probably due at least
in part to the i.p. route of administration for ascites.
Ertumaxomab is another triomab targeting HER2, which may be of use
for metastatic breast cancer. Bi20 is another triomab that targets
CD20. In vitro, Bi20 exhibited efficient lyis of B cells from CLL
patients (Chames & Baty, mAbs 1:539-47).
BITE.RTM. refers to tandem scFvs that are joined by a short peptide
linker (Chames & Baty, mAbs 1:539-47). Blinatumomab is an
anti-CD19.times.anti-CD3 BITE.RTM. with reported efficacy in
hematologic cancers, such as non-Hodgkin's lymphoma and ALL, at
very low concentrations (Nagorsen et al., 2009, Leukemia &
Lymphoma 50:886-91; Chames & Baty, mAbs 1:539-47; Topp et al.,
2012, Blood 120:5185-87; Bargou et al., 2008, Science 321:974-77).
Another BITE.RTM. with specificity for EpCAM has been used in
gastrointestinal, ovarian, colorectal and lung cancer (Amann et
al., 2009, J Immunother 32:452-64; Chames & Baty, mAbs
1:539-47). Another BITE.RTM. (MEDI-565) targeted to CEACAM5 has
been proposed for use in melanoma, colorectal, lung, pancreatic,
stomach, ovarian, uterine, and breast cancers (Sanders et al.,
1994, J Pathol 172:343-8). BITE.RTM. has been reported to exhibit
anti-tumor activity at picomolar or even femtomolar concentrations
(Chames & Baty, mAbs 1:539-47).
Another method of bsAb formation, involving assembly of two heavy
and two light chains derived from two different pre-existing
antibodies, is based on a knobs-into-holes approach that
facilitates heterodimer formation and prevents homodimer formation
(Schaefer et al., 2011, Proc Natl. Acad Sci USA 108:11187-92). The
"CrossMab" technique further involves the exchange of heavy and
light chain domains within the Fab of one half of the bispecific
antibody, making the two arms so different that light-heavy chain
mispairing can not occur (Schaefer et al., 2011). The
knobs-into-holes approach introduces amino acids with bulky side
chains into the CH3 domain of one heavy chain that fit into
appropriately designed cavities in the CH3 domain of the other
heavy chain. The combination of approaches prevents mis-match of
both heavy chain to heavy chain and heavy chain to light chain
interactions, resulting in primarily a single product. The initial
CrossMab, generated against angiopoietin-2 (Ang-2) and VEGF-A,
exhibited binding characteristics comparable to the parent mAbs,
with potent anti-angiogenic and anti-tumoral activity (Schaefer et
al., 2011, Proc Natl. Acad Sci USA 108:11187-92; Kienast et al.,
Clin Cancer Res, Oct. 25, 2013, Epub ahead of print).
In addition to the DART.TM. technology discussed above, other
approaches to bsAb production have included tetravalent IgG-scFv
fusions (Dong et al., 2011, MAbs 3:273-88); dual-acting Fab (DAF)
antibodies (Bostrom et al., 2009, Science 323:1610-14); Igg-like
dual-variable domain antibodies (DVD-Ig) (Wu et al., 2007, Nat
Biotechnol 25:1290-97); and use of dynamic exchange between IgG4
molecules (van der Neut Kolfschoten et al., 2007, Science
317:1554-57). Although the DNL.TM. technology discussed below is
preferred for formation of leukocyte redirecting bsAbs, the person
of ordinary skill will realize that other types of bsAbs may be
used in the claimed methods and compositions.
DOCK-AND-LOCK.TM. (DNL.TM.)
In some embodiments, a bispecific antibody, either alone or else
complexed to one or more effectors such as cytokines, is formed as
a DOCK-AND-LOCK.TM. (DNL.TM.) complex (see, e.g., U.S. Pat. Nos.
7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,901,680;
7,906,118; 7,981,398; 8,003,111, the Examples section of each of
which is incorporated herein by reference.) Generally, the
technique takes advantage of the specific and high-affinity binding
interactions that occur between a dimerization and docking domain
(DDD) sequence of the regulatory (R) subunits of cAMP-dependent
protein kinase (PKA) and an anchor domain (AD) sequence derived
from any of a variety of AKAP proteins (Baillie et al., FEBS
Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.
2004; 5: 959). The DDD and AD peptides may be attached to any
protein, peptide or other molecule. Because the DDD sequences
spontaneously dimerize and bind to the AD sequence, the technique
allows the formation of complexes between any selected molecules
that may be attached to DDD or AD sequences.
Although the standard DNL.TM. complex comprises a trimer with two
DDD-linked molecules attached to one AD-linked molecule, variations
in complex structure allow the formation of dimers, trimers,
tetramers, pentamers, hexamers and other multimers. In some
embodiments, the DNL.TM. complex may comprise two or more
antibodies, antibody fragments or fusion proteins which bind to the
same antigenic determinant or to two or more different antigens.
The DNL.TM. complex may also comprise one or more other effectors,
such as proteins, peptides, immunomodulators, cytokines,
interleukins, interferons, binding proteins, peptide ligands,
carrier proteins, toxins, ribonucleases such as onconase,
inhibitory oligonucleotides such as siRNA, antigens or
xenoantigens, polymers such as PEG, enzymes, therapeutic agents,
hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic
agents or any other molecule or aggregate.
PKA, which plays a central role in one of the best studied signal
transduction pathways triggered by the binding of the second
messenger cAMP to the R subunits, was first isolated from rabbit
skeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968;
243:3763). The structure of the holoenzyme consists of two
catalytic subunits held in an inactive form by the R subunits
(Taylor, J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found
with two types of R subunits (RI and RII), and each type has
.alpha. and .beta. isoforms (Scott, Pharmacol. Ther. 1991; 50:123).
Thus, the four isoforms of PKA regulatory subunits are RI.alpha.,
RI.beta., RII.alpha. and RII.beta., each of which comprises a DDD
moiety amino acid sequence. The R subunits have been isolated only
as stable dimers and the dimerization domain has been shown to
consist of the first 44 amino-terminal residues of RII.alpha.
(Newlon et al., Nat. Struct. Biol. 1999; 6:222). As discussed
below, similar portions of the amino acid sequences of other
regulatory subunits are involved in dimerization and docking, each
located near the N-terminal end of the regulatory subunit. Binding
of cAMP to the R subunits leads to the release of active catalytic
subunits for a broad spectrum of serine/threonine kinase
activities, which are oriented toward selected substrates through
the compartmentalization of PKA via its docking with AKAPs (Scott
et al., J. Biol. Chem. 1990; 265; 21561)
Since the first AKAP, microtubule-associated protein-2, was
characterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA
1984; 81:6723), more than 50 AKAPs that localize to various
sub-cellular sites, including plasma membrane, actin cytoskeleton,
nucleus, mitochondria, and endoplasmic reticulum, have been
identified with diverse structures in species ranging from yeast to
humans (Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The
AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr
et al., J. Biol. Chem. 1991; 266:14188). The amino acid sequences
of the AD are varied among individual AKAPs, with the binding
affinities reported for Rh dimers ranging from 2 to 90 nM (Alto et
al., Proc. Natl. Acad. Sci. USA 2003; 100:4445). AKAPs will only
bind to dimeric R subunits. For human RII.alpha., the AD binds to a
hydrophobic surface formed by the 23 amino-terminal residues
(Colledge and Scott, Trends Cell Biol. 1999; 6:216). Thus, the
dimerization domain and AKAP binding domain of human RII.alpha. are
both located within the same N-terminal 44 amino acid sequence
(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO
J. 2001; 20:1651), which is termed the DDD herein.
We have developed a platform technology to utilize the DDD of human
PKA regulatory subunits and the AD of AKAP as an excellent pair of
linker modules for docking any two entities, referred to hereafter
as A and B, into a noncovalent complex, which could be further
locked into a DNL.TM. complex through the introduction of cysteine
residues into both the DDD and AD at strategic positions to
facilitate the formation of disulfide bonds. The general
methodology of the approach is as follows. Entity A is constructed
by linking a DDD sequence to a precursor of A, resulting in a first
component hereafter referred to as a. Because the DDD sequence
would effect the spontaneous formation of a dimer, A would thus be
composed of a.sub.2. Entity B is constructed by linking an AD
sequence to a precursor of B, resulting in a second component
hereafter referred to as b. The dimeric motif of DDD contained in
a.sub.2 will create a docking site for binding to the AD sequence
contained in b, thus facilitating a ready association of a.sub.2
and b to form a binary, trimeric complex composed of a.sub.2b. This
binding event is stabilized with a subsequent reaction to
covalently secure the two entities via disulfide bridges, which
occurs very efficiently based on the principle of effective local
concentration because the initial binding interactions should bring
the reactive thiol groups placed onto both the DDD and AD into
proximity (Chmura et al., Proc. Natl. Acad. Sci. USA 2001; 98:8480)
to ligate site-specifically. Using various combinations of linkers,
adaptor modules and precursors, a wide variety of DNL.TM.
constructs of different stoichiometry may be produced and used
(see, e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866;
7,527,787 and 7,666,400.)
By attaching the DDD and AD away from the functional groups of the
two precursors, such site-specific ligations are also expected to
preserve the original activities of the two precursors. This
approach is modular in nature and potentially can be applied to
link, site-specifically and covalently, a wide range of substances,
including peptides, proteins, antibodies, antibody fragments, and
other effector moieties with a wide range of activities. Utilizing
the fusion protein method of constructing AD and DDD conjugated
effectors described in the Examples below, virtually any protein or
peptide may be incorporated into a DNL.TM. construct. However, the
technique is not limiting and other methods of conjugation may be
utilized.
A variety of methods are known for making fusion proteins,
including nucleic acid synthesis, hybridization and/or
amplification to produce a synthetic double-stranded nucleic acid
encoding a fusion protein of interest. Such double-stranded nucleic
acids may be inserted into expression vectors for fusion protein
production by standard molecular biology techniques (see, e.g.
Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2.sup.nd
Ed, 1989). In such preferred embodiments, the AD and/or DDD moiety
may be attached to either the N-terminal or C-terminal end of an
effector protein or peptide. However, the skilled artisan will
realize that the site of attachment of an AD or DDD moiety to an
effector moiety may vary, depending on the chemical nature of the
effector moiety and the part(s) of the effector moiety involved in
its physiological activity. Site-specific attachment of a variety
of effector moieties may be performed using techniques known in the
art, such as the use of bivalent cross-linking reagents and/or
other chemical conjugation techniques.
Dock-and-Lock.TM. (DNL.TM.) technology has been used to produce a
variety of complexes in assorted formats (Rossi et al., 2012,
Bioconjug Chem 23:309-23). Bispecific hexavalent antibodies
(bsHexAbs) based on veltuzumab (anti-CD20) and epratuzumab
(anti-CD22) were constructed by combining a stabilized (Fab).sub.2
fused to a dimerization and docking domain (DDD) with an IgG
containing an anchor domain (AD) appended at the C-terminus of each
heavy chain (C.sub.H3-AD2-IgG) (Rossi et al., 2009, Blood 113,
6161-71). Compared to mixtures of their parental mAbs, these
Fc-based bsHexAbs, referred to henceforth as "Fc-bsHexAbs", induced
unique signaling events (Gupta et al., 2010, Blood 116:3258-67),
and exhibited potent cytotoxicity in vitro. However, the
Fc-bsHexAbs were cleared from circulation of mice approximately
twice as fast as the parental mAbs (Rossi et al., 2009, Blood 113,
6161-71). Although the Fc-bsHexAbs are highly stable ex vivo, it is
possible that some dissociation occurs in vivo, for example by
intracellular processing. Further, the Fc-bsHexAbs lack CDC
activity.
Fc-based immunocytokines have also been assembled as DNL.TM.
complexes, comprising two or four molecules of interferon-alpha 2b
(IFN.alpha.2b) fused to the C-terminal end of the C.sub.H3-AD2-IgG
Fc (Rossi et al., 2009, Blood 114:3864-71; Rossi et al., 2010,
Cancer Res 70:7600-09; Rossi et al., 2011, Blood 118:1877-84). The
Fc-IgG-IFN.alpha. maintained high specific activity, approaching
that of recombinant IFN.alpha., and were remarkably potent in vitro
and in vivo against non-Hodgkin lymphoma (NHL) xenografts. The
T.sub.1/2 of the Fc-IgG-IFN.alpha. in mice was longer than
PEGylated IFN.alpha., but half as long as the parental mAbs.
Similar to the Fc-bsHexAbs, the Fc-IgG-IFN.alpha. dissociated in
vivo over time and exhibited diminished CDC, but ADCC was
enhanced.
Structure-Function Relationships in AD and DDD Moieties
For different types of DNL.TM. constructs, different AD or DDD
sequences may be utilized. Exemplary DDD and AD sequences are
provided below.
TABLE-US-00003 DDD1 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2 (SEQ ID NO: 2)
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1 (SEQ ID NO: 3)
QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4) CGQIEYLAKQIVDNAIQQAGC
The skilled artisan will realize that DDD1 and DDD2 are based on
the DDD sequence of the human RII.alpha. isoform of protein kinase
A. However, in alternative embodiments, the DDD and AD moieties may
be based on the DDD sequence of the human RI.alpha. form of protein
kinase A and a corresponding AKAP sequence, as exemplified in DDD3,
DDD3C and AD3 below.
TABLE-US-00004 DDD3 (SEQ ID NO: 5)
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK DDD3C (SEQ ID
NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLE KEEAK AD3
(SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC
In other alternative embodiments, other sequence variants of AD
and/or DDD moieties may be utilized in construction of the DNL.TM.
complexes. For example, there are only four variants of human PKA
DDD sequences, corresponding to the DDD moieties of PKA RI.alpha.,
RII.alpha., RI.beta. and RII.beta.. The RII.alpha. DDD sequence is
the basis of DDD1 and DDD2 disclosed above. The four human PKA DDD
sequences are shown below. The DDD sequence represents residues
1-44 of RII.alpha., 1-44 of RII.beta., 12-61 of RI.alpha. and 13-66
of RI.beta.. (Note that the sequence of DDD1 is modified slightly
from the human PKA RII.alpha. DDD moiety.)
TABLE-US-00005 PKA RI.alpha. (SEQ ID NO: 8)
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RI.beta.
(SEQ ID NO: 9) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR
QILA PKA RII.alpha. (SEQ ID NO: 10)
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RII.beta. (SEQ ID
NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER
The structure-function relationships of the AD and DDD domains have
been the subject of investigation. (See, e.g., Burns-Hamuro et al.,
2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem
276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA
100:4445-50; Hundsrucker et al., 2006, Biochem J 396:297-306;
Stokka et al., 2006, Biochem J 400:493-99; Gold et al., 2006, Mol
Cell 24:383-95; Kinderman et al., 2006, Mol Cell 24:397-408, the
entire text of each of which is incorporated herein by
reference.)
For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined
the crystal structure of the AD-DDD binding interaction and
concluded that the human DDD sequence contained a number of
conserved amino acid residues that were important in either dimer
formation or AKAP binding, underlined in SEQ ID NO:1 below. (See
FIG. 1 of Kinderman et al., 2006, incorporated herein by
reference.) The skilled artisan will realize that in designing
sequence variants of the DDD sequence, one would desirably avoid
changing any of the underlined residues, while conservative amino
acid substitutions might be made for residues that are less
critical for dimerization and AKAP binding.
TABLE-US-00006 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
As discussed in more detail below, conservative amino acid
substitutions have been characterized for each of the twenty common
L-amino acids. Thus, based on the data of Kinderman (2006) and
conservative amino acid substitutions, potential alternative DDD
sequences based on SEQ ID NO:1 are shown in Table 2. In devising
Table 2, only highly conservative amino acid substitutions were
considered. For example, charged residues were only substituted for
residues of the same charge, residues with small side chains were
substituted with residues of similar size, hydroxyl side chains
were only substituted with other hydroxyls, etc. Because of the
unique effect of proline on amino acid secondary structure, no
other residues were substituted for proline. A limited number of
such potential alternative DDD moiety sequences are shown in SEQ ID
NO:12 to SEQ ID NO:31 below. The skilled artisan will realize that
alternative species within the genus of DDD moieties can be
constructed by standard techniques, for example using a commercial
peptide synthesizer or well known site-directed mutagenesis
techniques. The effect of the amino acid substitutions on AD moiety
binding may also be readily determined by standard binding assays,
for example as disclosed in Alto et al. (2003, Proc Natl Acad Sci
USA 100:4445-50).
TABLE-US-00007 TABLE 2 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 87.
S H I Q I P P G L T E L L Q G Y T V E V L R T K N A S D N A S D K R
Q Q P P D L V E F A V E Y F T R L R E A R A N N E D L D S K K D L K
L I I I V V V THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO: 12) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:
13) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)
SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)
SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)
SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)
SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)
SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)
SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)
SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)
SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)
SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)
SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)
SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)
SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)
Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed a
bioinformatic analysis of the AD sequence of various AKAP proteins
to design an RII selective AD sequence called AKAP-IS (SEQ ID
NO:3), with a binding constant for DDD of 0.4 nM. The AKAP-IS
sequence was designed as a peptide antagonist of AKAP binding to
PKA. Residues in the AKAP-IS sequence where substitutions tended to
decrease binding to DDD are underlined in SEQ ID NO:3 below. The
skilled artisan will realize that in designing sequence variants of
the AD sequence, one would desirably avoid changing any of the
underlined residues, while conservative amino acid substitutions
might be made for residues that are less critical for DDD binding.
Table 3 shows potential conservative amino acid substitutions in
the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar to that shown
for DDD1 (SEQ ID NO:1) in Table 2 above.
A limited number of such potential alternative AD moiety sequences
are shown in SEQ ID NO:32 to SEQ ID NO:49 below. Other species
within the genus of possible AD moiety sequences could be made,
tested and used by the skilled artisan, based on the data of Alto
et al. (2003). It is noted that FIG. 2 of Alto (2003) shows a
number of amino acid substitutions that may be made, while
retaining binding activity to DDD moieties, based on actual binding
experiments.
TABLE-US-00008 AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA
TABLE-US-00009 TABLE 3 Conservative Amino Acid Substitutions in AD1
(SEQ ID NO: 3). Consensus sequence disclosed as SEQ ID NO: 88. Q I
E Y L A K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S V
NIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)
QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)
QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)
QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)
QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)
QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)
QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)
QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)
QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO:
49)
Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography and
peptide screening to develop a SuperAKAP-IS sequence (SEQ ID
NO:50), exhibiting a five order of magnitude higher selectivity for
the RII isoform of PKA compared with the RI isoform. Underlined
residues indicate the positions of amino acid substitutions,
relative to the AKAP-IS sequence, which increased binding to the
DDD moiety of RII.alpha.. In this sequence, the N-terminal Q
residue is numbered as residue number 4 and the C-terminal A
residue is residue number 20. Residues where substitutions could be
made to affect the affinity for RII.alpha. were residues 8, 11, 15,
16, 18, 19 and 20 (Gold et al., 2006). It is contemplated that in
certain alternative embodiments, the SuperAKAP-IS sequence may be
substituted for the AKAP-IS AD moiety sequence to prepare DNL.TM.
constructs. Other alternative sequences that might be substituted
for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.
Substitutions relative to the AKAP-IS sequence are underlined. It
is anticipated that, as with the AD2 sequence shown in SEQ ID NO:4,
the AD moiety may also include the additional N-terminal residues
cysteine and glycine and C-terminal residues glycine and
cysteine.
TABLE-US-00010 SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQA
Alternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA (SEQ
ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA
FIG. 2 of Gold et al. disclosed additional DDD-binding sequences
from a variety of AKAP proteins, shown below.
TABLE-US-00011 RII-Specific AKAPs AKAP-KL (SEQ ID NO: 54)
PLEYQAGLLVQNAIQQAI AKAP79 (SEQ ID NO: 55) LLIETASSLVKNAIQLSI
AKAP-Lbc (SEQ ID NO: 56) LIEEAASRIVDAVIEQVK RI-Specific AKAPs
AKAPce (SEQ ID NO: 57) alyqfadrfselviseal riad (SEQ ID NO: 58)
leqvanqladqiikeat pv38 (SEQ ID NO: 59) feelawkiakmiwsdvf
Dual-Specificity AKAPs akap7 (SEQ ID NO: 60) elvrlskrlvenavlkav
map2d (SEQ ID NO: 61) taeevsarivqvvtaeav dakap1 (SEQ ID NO: 62)
qikqaafqlisqvileat Dakap2 (SEQ ID NO: 63) lawkiakmivsdvmqq
Stokka et al. (2006, Biochem J 400:493-99) also developed peptide
competitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. The
peptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD
(SEQ ID NO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide
exhibited a greater affinity for the RII isoform of PKA, while the
RIAD and PV-38 showed higher affinity for RI.
TABLE-US-00012 Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD
(SEQ ID NO: 65) LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66)
FEELAWKIAKMIWSDVFQQC
Hundsrucker et al. (2006, Biochem J 396:297-306) developed still
other peptide competitors for AKAP binding to PKA, with a binding
constant as low as 0.4 nM to the DDD of the RII form of PKA. The
sequences of various AKAP antagonistic peptides are provided in
Table 1 of Hundsrucker et al., reproduced in Table 4 below. AKAPIS
represents a synthetic RII subunit-binding peptide. All other
peptides are derived from the RII-binding domains of the indicated
AKAPs.
TABLE-US-00013 TABLE 4 AKAP Peptide sequences Peptide Sequence
AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-P QIEYLAKQIPDNAIQQA
(SEQ ID NO: 67) Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69)
AKAP7.delta.-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70)
AKAP7.delta.-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71)
AKAP7.delta.-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72)
AKAP7.delta.-P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73)
AKAP7.delta.-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74)
AKAP7.delta.-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pep
LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pep
QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pep
LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pep
NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pep
VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pep
NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pep
TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pep
ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)
Residues that were highly conserved among the AD domains of
different AKAP proteins are indicated below by underlining with
reference to the AKAP IS sequence (SEQ ID NO:3). The residues are
the same as observed by Alto et al. (2003), with the addition of
the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al.
(2006), incorporated herein by reference.) The sequences of peptide
antagonists with particularly high affinities for the RII DDD
sequence were those of AKAP-IS, AKAP7.delta.-wt-pep,
AKAP7.delta.-L304T-pep and AKAP7.delta.-L308D-pep.
TABLE-US-00014 AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA
Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree of
sequence homology between different AKAP-binding DDD sequences from
human and non-human proteins and identified residues in the DDD
sequences that appeared to be the most highly conserved among
different DDD moieties. These are indicated below by underlining
with reference to the human PKA RII.alpha. DDD sequence of SEQ ID
NO:1. Residues that were particularly conserved are further
indicated by italics. The residues overlap with, but are not
identical to those suggested by Kinderman et al. (2006) to be
important for binding to AKAP proteins. The skilled artisan will
realize that in designing sequence variants of DDD, it would be
most preferred to avoid changing the most conserved residues
(italicized), and it would be preferred to also avoid changing the
conserved residues (underlined), while conservative amino acid
substitutions may be considered for residues that are neither
underlined nor italicized.
TABLE-US-00015 (SEQ ID NO: 1)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
A modified set of conservative amino acid substitutions for the
DDD1 (SEQ ID NO:1) sequence, based on the data of Carr et al.
(2001) is shown in Table 5. Even with this reduced set of
substituted sequences, there are over 65,000 possible alternative
DDD moiety sequences that may be produced, tested and used by the
skilled artisan without undue experimentation. The skilled artisan
could readily derive such alternative DDD amino acid sequences as
disclosed above for Table 2 and Table 3.
TABLE-US-00016 TABLE 5 Conservative Amino Acid Substitutions in
DDD1 (SEQ ID NO: 1). Consensus sequence disclosed as SEQ ID NO: 89.
S H I Q I P P G L T E L L Q G Y T V E V L R T N S I L A Q Q P P D L
V E F A V E Y F T R L R E A R A N I D S K K L L L I I A V V
The skilled artisan will realize that these and other amino acid
substitutions in the DDD or AD amino acid sequences may be utilized
to produce alternative species within the genus of AD or DDD
moieties, using techniques that are standard in the field and only
routine experimentation.
Amino Acid Substitutions
In alternative embodiments, the disclosed methods and compositions
may involve production and use of proteins or peptides with one or
more substituted amino acid residues. For example, the DDD and/or
AD sequences used to make DNL.TM. constructs may be modified as
discussed above.
The skilled artisan will be aware that, in general, amino acid
substitutions typically involve the replacement of an amino acid
with another amino acid of relatively similar properties (i.e.,
conservative amino acid substitutions). The properties of the
various amino acids and effect of amino acid substitution on
protein structure and function have been the subject of extensive
study and knowledge in the art.
For example, the hydropathic index of amino acids may be considered
(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The
relative hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules. Each amino
acid has been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics (Kyte & Doolittle,
1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);
alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine
(-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);
asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making
conservative substitutions, the use of amino acids whose
hydropathic indices are within .+-.2 is preferred, within .+-.1 are
more preferred, and within .+-.0.5 are even more preferred.
Amino acid substitution may also take into account the
hydrophilicity of the amino acid residue (e.g., U.S. Pat. No.
4,554,101). Hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0);
glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1);
alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine
(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine
(-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of
amino acids with others of similar hydrophilicity is preferred.
Other considerations include the size of the amino acid side chain.
For example, it would generally not be preferred to replace an
amino acid with a compact side chain, such as glycine or serine,
with an amino acid with a bulky side chain, e.g., tryptophan or
tyrosine. The effect of various amino acid residues on protein
secondary structure is also a consideration. Through empirical
study, the effect of different amino acid residues on the tendency
of protein domains to adopt an alpha-helical, beta-sheet or reverse
turn secondary structure has been determined and is known in the
art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245;
1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J.,
26:367-384).
Based on such considerations and extensive empirical study, tables
of conservative amino acid substitutions have been constructed and
are known in the art. For example: arginine and lysine; glutamate
and aspartate; serine and threonine; glutamine and asparagine; and
valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile,
val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp
(D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp;
Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala,
phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg;
Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P)
ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe,
thr, ser; Val (V) ile, leu, met, phe, ala.
Other considerations for amino acid substitutions include whether
or not the residue is located in the interior of a protein or is
solvent exposed. For interior residues, conservative substitutions
would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala;
Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met;
Phe and Tyr; Tyr and Trp. (See, e.g., PROWL website at
rockefeller.edu) For solvent exposed residues, conservative
substitutions would include: Asp and Asn; Asp and Glu; Glu and Gln;
Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser;
Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile;
Ile and Val; Phe and Tyr. (Id.) Various matrices have been
constructed to assist in selection of amino acid substitutions,
such as the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix,
McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix,
Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler
matrix (Idem.)
In determining amino acid substitutions, one may also consider the
existence of intermolecular or intramolecular bonds, such as
formation of ionic bonds (salt bridges) between positively charged
residues (e.g., His, Arg, Lys) and negatively charged residues
(e.g., Asp, Glu) or disulfide bonds between nearby cysteine
residues.
Methods of substituting any amino acid for any other amino acid in
an encoded protein sequence are well known and a matter of routine
experimentation for the skilled artisan, for example by the
technique of site-directed mutagenesis or by synthesis and assembly
of oligonucleotides encoding an amino acid substitution and
splicing into an expression vector construct.
Therapeutic Agents
In alternative embodiments, therapeutic agents such as cytotoxic
agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics,
hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins,
enzymes or other agents may be used, either conjugated to the
subject bsAbs, ADCs and/or antibodies or separately administered
before, simultaneously with, or after the bsAbs, ADCs and/or
antibodies. Drugs of use may possess a pharmaceutical property
selected from the group consisting of antimitotic, antikinase,
alkylating, antimetabolite, antibiotic, alkaloid, anti-angiogenic,
pro-apoptotic agents and combinations thereof.
Exemplary drugs of use may include, but are not limited to,
5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,
anthracyclines, axitinib, AVL-101, AVL-291, bendamustine,
bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan,
calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,
carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2
inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine,
camptothecans, crizotinib, cyclophosphamide, cytarabine,
dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,
daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin
glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,
entinostat, estrogen receptor binding agents, etoposide (VP16),
etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,
floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine,
flutamide, farnesyl-protein transferase inhibitors, flavopiridol,
fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,
gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,
ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide,
leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,
nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel,
PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib,
streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an
aqueous form of DTIC), transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vatalanib,
vinorelbine, vinblastine, vincristine, vinca alkaloids and
ZD1839.
Toxins of use may include ricin, abrin, alpha toxin, saporin,
ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta
and IP-10.
In certain embodiments, anti-angiogenic agents, such as
angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies,
anti-PlGF peptides and antibodies, anti-vascular growth factor
antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and
peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF
(macrophage migration-inhibitory factor) antibodies, laminin
peptides, fibronectin peptides, plasminogen activator inhibitors,
tissue metalloproteinase inhibitors, interferons, interleukin-12,
IP-10, Gro-.beta., thrombospondin, 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin-2,
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide (roquinimex), thalidomide, pentoxifylline, genistein,
TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir,
vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline
may be of use.
Immunomodulators of use may be selected from a cytokine, a stem
cell growth factor, a lymphotoxin, a hematopoietic factor, a colony
stimulating factor (CSF), an interferon (IFN), erythropoietin,
thrombopoietin and a combination thereof. Specifically useful are
lymphotoxins such as tumor necrosis factor (TNF), hematopoietic
factors, such as interleukin (IL), colony stimulating factor, such
as granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon, such as
interferons-.alpha., -.beta. or -.lamda., and stem cell growth
factor, such as that designated "S1 factor". Included among the
cytokines are growth hormones such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
prorelaxin; glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); hepatic growth factor; prostaglandin, fibroblast
growth factor; prolactin; placental lactogen, OB protein; tumor
necrosis factor-.alpha. and -.beta.; mullerian-inhibiting
substance; mouse gonadotropin-associated peptide; inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve growth factors such as NGF-.beta.; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -.beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF); interleukins (ILs)
such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin,
thrombospondin, endostatin, tumor necrosis factor and LT.
Radionuclides of use include, but are not limited to--.sup.111In,
.sup.177Lu, .sup.212Bi, .sup.213Bi, .sup.211At, .sup.62Cu,
.sup.67Cu, .sup.90Y, .sup.125I, .sup.131I, .sup.32P, .sup.33P,
.sup.47Sc, .sup.111Ag, .sup.67Ga, .sup.142Pr, .sup.153Sm,
.sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.186Re, .sup.188Re,
.sup.189Re, .sup.212Pb, .sup.223Ra, .sup.225Ac, .sup.59Fe,
.sup.75Se, .sup.77As, .sup.89Sr, .sup.99Mo, .sup.105Rh, .sup.109Pd,
.sup.143Pr, .sup.149Pm, .sup.169Er, .sup.194Ir, .sup.198Au,
.sup.199Au, .sup.211Pb, and .sup.227Th. The therapeutic
radionuclide preferably has a decay-energy in the range of 20 to
6,000 keV, preferably in the ranges 60 to 200 keV for an Auger
emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for
an alpha emitter. Maximum decay energies of useful
beta-particle-emitting nuclides are preferably 20-5,000 keV, more
preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also
preferred are radionuclides that substantially decay with
Auger-emitting particles. For example, Co-58, Ga-67, Br-80m,
Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m and
Ir-192. Decay energies of useful beta-particle-emitting nuclides
are preferably <1,000 keV, more preferably <100 keV, and most
preferably <70 keV. Also preferred are radionuclides that
substantially decay with generation of alpha-particles. Such
radionuclides include, but are not limited to: Dy-152, At-211,
Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217,
Bi-213, Th-227 and Fm-255. Decay energies of useful
alpha-particle-emitting radionuclides are preferably 2,000-10,000
keV, more preferably 3,000-8,000 keV, and most preferably
4,000-7,000 keV. Additional potential radioisotopes of use include
.sup.11C, .sup.13N, .sup.15O, .sup.75Br, .sup.198Au, .sup.224Ac,
.sup.126I, .sup.133I, .sup.77Br, .sup.113mIn, .sup.95Ru, .sup.97Ru,
.sup.103Ru, .sup.105Ru, .sup.107Hg, .sup.203Hg, .sup.121mTe,
.sup.122mTe, .sup.125mTe, .sup.165Tm, .sup.167Tm, .sup.168Tm,
.sup.197Pt, .sup.109Pd, .sup.105Rh, .sup.142Pr, .sup.143Pr,
.sup.161Tb, .sup.166Ho, .sup.199Au, .sup.57Co, .sup.58Co,
.sup.51Cr, .sup.59Fe, .sup.75Se, .sup.201Tl, .sup.225Ac, .sup.76Br,
.sup.169Yb, and the like. Some useful diagnostic nuclides may
include .sup.18F, .sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu,
.sup.67Ga, .sup.68Ga, .sup.86Y, .sup.89Zr, .sup.94Tc, .sup.94mTc,
.sup.99mTc, or, .sup.111In.
Therapeutic agents may include a photoactive agent or dye.
Fluorescent compositions, such as fluorochrome, and other
chromogens, or dyes, such as porphyrins sensitive to visible light,
have been used to detect and to treat lesions by directing the
suitable light to the lesion. In therapy, this has been termed
photoradiation, phototherapy, or photodynamic therapy. See Joni et
al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES
(Libreria Progetto 1985); van den Bergh, Chem. Britain (1986),
22:430. Moreover, monoclonal antibodies have been coupled with
photoactivated dyes for achieving phototherapy. See Mew et al., J.
Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;
Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,
Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin.
Biol. Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med.
(1989), 9:422; Pelegrin et al., Cancer (1991), 67:2529.
Other useful therapeutic agents may comprise oligonucleotides,
especially antisense oligonucleotides that preferably are directed
against oncogenes and oncogene products, such as bcl-2 or p53. A
preferred form of therapeutic oligonucleotide is siRNA. The skilled
artisan will realize that any siRNA or interference RNA species may
be attached to an antibody or fragment thereof for delivery to a
targeted tissue. Many siRNA species against a wide variety of
targets are known in the art, and any such known siRNA may be
utilized in the claimed methods and compositions.
Known siRNA species of potential use include those specific for
IKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR
(U.S. Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453);
CDC20 (U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S.
Pat. No. 7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic
anhydrase II (U.S. Pat. No. 7,579,457); complement component 3
(U.S. Pat. No. 7,582,746); interleukin-1 receptor-associated kinase
4 (IRAK4) (U.S. Pat. No. 7,592,443); survivin (U.S. Pat. No.
7,608,7070); superoxide dismutase 1 (U.S. Pat. No. 7,632,938); MET
proto-oncogene (U.S. Pat. No. 7,632,939); amyloid beta precursor
protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R (U.S. Pat. No.
7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complement factor B
(U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), and
apolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of
each referenced patent incorporated herein by reference.
Additional siRNA species are available from known commercial
sources, such as Sigma-Aldrich (St Louis, Mo.), Invitrogen
(Carlsbad, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.),
Ambion (Austin, Tex.), Dharmacon (Thermo Scientific, Lafayette,
Colo.), Promega (Madison, Wis.), Minis Bio (Madison, Wis.) and
Qiagen (Valencia, Calif.), among many others. Other publicly
available sources of siRNA species include the siRNAdb database at
the Stockholm Bioinformatics Centre, the MIT/ICBP siRNA Database,
the RNAi Consortium shRNA Library at the Broad Institute, and the
Probe database at NCBI. For example, there are 30,852 siRNA species
in the NCBI Probe database. The skilled artisan will realize that
for any gene of interest, either a siRNA species has already been
designed, or one may readily be designed using publicly available
software tools. Any such siRNA species may be delivered using the
subject DNL.TM. complexes.
Methods of Therapeutic Treatment
Various embodiments concern methods of treating a cancer in a
subject, such as a mammal, including humans, domestic or companion
pets, such as dogs and cats, comprising administering to the
subject a therapeutically effective amount of a combination of
cytotoxic and/or immunomodulatory agents.
The administration of the cytotoxic bsAbs, ADCs and/or checkpoint
inhibitor antibodies can be supplemented by administering
concurrently or sequentially a therapeutically effective amount of
another antibody that binds to or is reactive with another antigen
on the surface of the target cell. Preferred additional MAbs
comprise at least one humanized, chimeric or human MAb selected
from the group consisting of a MAb reactive with CD4, CD5, CD8,
CD14, CD15, CD16, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD30,
CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70,
CD74, CD79a, CD79b, CD80, CD95, CD126, CD133, CD138, CD154,
CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase
IX, PAM4 antigen, MUC1, MUC2, MUC3, MUC4, MUC5, Ia, MIF, HM1.24,
HLA-DR, tenascin, Flt-3, VEGFR, PlGF, ILGF, IL-6, IL-25, tenascin,
TRAIL-R1, TRAIL-R2, complement factor C5, oncogene product, or a
combination thereof. Various antibodies of use, such as anti-CD19,
anti-CD20, and anti-CD22 antibodies, are known to those of skill in
the art. See, for example, Ghetie et al., Cancer Res. 48:2610
(1988); Hekman et al., Cancer Immunol. Immunother. 32:364 (1991);
Longo, Curr. Opin. Oncol. 8:353 (1996), U.S. Pat. Nos. 5,798,554;
6,187,287; 6,306,393; 6,676,924; 7,109,304; 7,151,164; 7,230,084;
7,230,085; 7,238,785; 7,238,786; 7,282,567; 7,300,655; 7,312,318;
7,501,498; 7,612,180; 7,670,804; and U.S. Patent Application Publ.
Nos. 20080131363; 20070172920; 20060193865; and 20080138333, the
Examples section of each incorporated herein by reference.
The combination therapy can be further supplemented with the
administration, either concurrently or sequentially, of at least
one therapeutic agent. For example, "CVB" (1.5 g/m.sup.2
cyclophosphamide, 200-400 mg/m.sup.2 etoposide, and 150-200
mg/m.sup.2 carmustine) is a regimen used to treat non-Hodgkin's
lymphoma. Patti et al., Eur. J. Haematol. 51: 18 (1993). Other
suitable combination chemotherapeutic regimens are well-known to
those of skill in the art. See, for example, Freedman et al.,
"Non-Hodgkin's Lymphomas," in CANCER MEDICINE, VOLUME 2, 3rd
Edition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger
1993). As an illustration, first generation chemotherapeutic
regimens for treatment of intermediate-grade non-Hodgkin's lymphoma
(NHL) include C-MOPP (cyclophosphamide, vincristine, procarbazine
and prednisone) and CHOP (cyclophosphamide, doxorubicin,
vincristine, and prednisone). A useful second generation
chemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,
doxorubicin, cyclophosphamide, vincristine, dexamethasone and
leucovorin), while a suitable third generation regimen is MACOP-B
(methotrexate, doxorubicin, cyclophosphamide, vincristine,
prednisone, bleomycin and leucovorin). Additional useful drugs
include phenyl butyrate, bendamustine, and bryostatin-1.
The combinations of therapeutic agents can be formulated according
to known methods to prepare pharmaceutically useful compositions,
whereby the bsAb, ADC, interferon and/or checkpoint inhibitor
antibody is combined in a mixture with a pharmaceutically suitable
excipient. Sterile phosphate-buffered saline is one example of a
pharmaceutically suitable excipient. Other suitable excipients are
well-known to those in the art. See, for example, Ansel et al.,
PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition
(Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company
1990), and revised editions thereof.
The subject bsAbs, ADCs, interferons and/or antibodies can be
formulated for intravenous administration via, for example, bolus
injection or continuous infusion. Preferably, the bsAb, ADC and/or
antibody is infused over a period of less than about 4 hours, and
more preferably, over a period of less than about 3 hours. For
example, the first bolus could be infused within 30 minutes,
preferably even 15 min, and the remainder infused over the next 2-3
hrs. Formulations for injection can be presented in unit dosage
form, e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions can take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and can contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient can be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
Additional pharmaceutical methods may be employed to control the
duration of action of the therapeutic combinations. Control release
preparations can be prepared through the use of polymers to complex
or adsorb the agents to be administered. For example, biocompatible
polymers include matrices of poly(ethylene-co-vinyl acetate) and
matrices of a polyanhydride copolymer of a stearic acid dimer and
sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The
rate of release from such a matrix depends upon the molecular
weight of the therapeutic agent, the amount of agent within the
matrix, and the size of dispersed particles. Saltzman et al.,
Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid
dosage forms are described in Ansel et al., PHARMACEUTICAL DOSAGE
FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger
1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th
Edition (Mack Publishing Company 1990), and revised editions
thereof.
The bsAbs, interferons and/or checkpoint inhibitor antibodies may
be administered to a mammal subcutaneously or even by other
parenteral routes, such as intravenously, intramuscularly,
intraperitoneally or intravascularly. ADCs may be administered
intravenously, intraperitoneally or intravascularly. Moreover, the
administration may be by continuous infusion or by single or
multiple boluses. Preferably, the bsAb, ADC, interferon and/or
checkpoint inhibitor antibody is infused over a period of less than
about 4 hours, and more preferably, over a period of less than
about 3 hours.
More generally, the dosage of an administered bsAb, ADC, interferon
and/or checkpoint inhibitor antibody for humans will vary depending
upon such factors as the patient's age, weight, height, sex,
general medical condition and previous medical history. It may be
desirable to provide the recipient with a dosage of bsAb, ADC
and/or antibody that is in the range of from about 1 mg/kg to 25
mg/kg as a single intravenous infusion, although a lower or higher
dosage also may be administered as circumstances dictate. A dosage
of 1-20 mg/kg for a 70 kg patient, for example, is 70-1,400 mg, or
41-824 mg/m.sup.2 for a 1.7-m patient. The dosage may be repeated
as needed, for example, once per week for 4-10 weeks, once per week
for 8 weeks, or once per week for 4 weeks. It may also be given
less frequently, such as every other week for several months, or
monthly or quarterly for many months, as needed in a maintenance
therapy.
Alternatively, a bsAb, ADC, and/or checkpoint inhibitor antibody
may be administered as one dosage every 2 or 3 weeks, repeated for
a total of at least 3 dosages. Or, the combination may be
administered twice per week for 4-6 weeks. If the dosage is lowered
to approximately 200-300 mg/m.sup.2 (340 mg per dosage for a 1.7-m
patient, or 4.9 mg/kg for a 70 kg patient), it may be administered
once or even twice weekly for 4 to 10 weeks. Alternatively, the
dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3
months. It has been determined, however, that even higher doses,
such as 20 mg/kg once weekly or once every 2-3 weeks can be
administered by slow i.v. infusion, for repeated dosing cycles. The
dosing schedule can optionally be repeated at other intervals and
dosage may be given through various parenteral routes, with
appropriate adjustment of the dose and schedule.
The person of ordinary skill will realize that while the dosage
schedules discussed above are relevant for ADCs, bsAbs and/or mAbs,
the interferon agents should be administered at substantially lower
dosages to avoid systemic toxicity. Dosages of interferons (such as
PEGINTERFERON) for humans are more typically in the microgram
range, for example 180 .mu.g s.c. once per week, or 100 to 180
.mu.g, or 135 .mu.g, or 135 .mu.g/1.73 m.sup.2, or 90 .mu.g/1.73
m.sup.2, or 250 .mu.g s.c. every other day may be of use, depending
on the type of interferon.
While the bsAbs, interferons, ADCs and/or checkpoint inhibitor
antibodies may be administered as a periodic bolus injection, in
alternative embodiments the bsAbs, ADCs, interferons and/or
checkpoint inhibitor antibodies may be administered by continuous
infusion. In order to increase the Cmax and extend the PK of the
therapeutic agents in the blood, a continuous infusion may be
administered for example by indwelling catheter. Such devices are
known in the art, such as HICKMAN.RTM., BROVIAC.RTM. or
PORT-A-CATH.RTM. catheters (see, e.g., Skolnik et al., Ther Drug
Monit 32:741-48, 2010) and any such known indwelling catheter may
be used. A variety of continuous infusion pumps are also known in
the art and any such known infusion pump may be used. The dosage
range for continuous infusion may be between 0.1 and 3.0 mg/kg per
day. More preferably, the bsAbs, ADCs, interferons and/or
checkpoint inhibitor antibodies can be administered by intravenous
infusions over relatively short periods of 2 to 5 hours, more
preferably 2-3 hours.
In preferred embodiments, the combination of agents is of use for
therapy of cancer. Examples of cancers include, but are not limited
to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and
leukemia, myeloma, or lymphoid malignancies. More particular
examples of such cancers are noted below and include: squamous cell
cancer (e.g., epithelial squamous cell cancer), Ewing sarcoma,
Wilms tumor, astrocytomas, lung cancer including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung and
squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma
multiforme, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors,
medullary thyroid cancer, differentiated thyroid carcinoma, breast
cancer, ovarian cancer, colon cancer, rectal cancer, endometrial
cancer or uterine carcinoma, salivary gland carcinoma, kidney or
renal cancer, prostate cancer, vulvar cancer, anal carcinoma,
penile carcinoma, as well as head-and-neck cancer. The term
"cancer" includes primary malignant cells or tumors (e.g., those
whose cells have not migrated to sites in the subject's body other
than the site of the original malignancy or tumor) and secondary
malignant cells or tumors (e.g., those arising from metastasis, the
migration of malignant cells or tumor cells to secondary sites that
are different from the site of the original tumor). Cancers
conducive to treatment methods of the present invention involves
cells which express, over-express, or abnormally express
IGF-1R.
Other examples of cancers or malignancies include, but are not
limited to: Acute Childhood Lymphoblastic Leukemia, Acute
Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid
Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular
Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Lymphoma,
Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult
Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related
Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile
Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain
Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter,
Central Nervous System (Primary) Lymphoma, Central Nervous System
Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical
Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood
(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,
Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma,
Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma,
Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's
Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and
Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood
Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal
and Supratentorial Primitive Neuroectodermal Tumors, Childhood
Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft
Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma,
Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon
Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell
Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer,
Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine
Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ
Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female
Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric
Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors,
Germ Cell Tumors, Gestational TROPhoblastic Tumor, Hairy Cell
Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's
Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal
Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell
Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal
Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,
Lymphoproliferative Disorders, Macroglobulinemia, Male Breast
Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma,
Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck
Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic
Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma
Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia,
Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and
Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,
Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell
Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer,
Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma,
Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant
Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian
Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic
Cancer, Paraproteinemias, Polycythemia vera, Parathyroid Cancer,
Penile Cancer, Pheochromocytoma, Pituitary Tumor, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, TROPhoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer,
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
The methods and compositions described and claimed herein may be
used to treat malignant or premalignant conditions and to prevent
progression to a neoplastic or malignant state, including but not
limited to those disorders described above. Such uses are indicated
in conditions known or suspected of preceding progression to
neoplasia or cancer, in particular, where non-neoplastic cell
growth consisting of hyperplasia, metaplasia, or most particularly,
dysplasia has occurred (for review of such abnormal growth
conditions, see Robbins and Angell, BASIC PATHOLOGY, 2d Ed., W. B.
Saunders Co., Philadelphia, pp. 68-79 (1976)).
Dysplasia is frequently a forerunner of cancer, and is found mainly
in the epithelia. It is the most disorderly form of non-neoplastic
cell growth, involving a loss in individual cell uniformity and in
the architectural orientation of cells. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation. Dysplastic disorders which can be treated include,
but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia,
cleidocranial dysplasia, congenital ectodermal dysplasia,
craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia,
fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic
dysplasia, mammary dysplasia, mandibulofacial dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous
dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculovertebral dysplasia, odontogenic dysplasia,
opthalmomandibulomelic dysplasia, periapical cemental dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial
dysplasia.
Additional pre-neoplastic disorders which can be treated include,
but are not limited to, benign dysproliferative disorders (e.g.,
benign tumors, fibrocystic conditions, tissue hypertrophy,
intestinal polyps or adenomas, and esophageal dysplasia),
leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar
cheilitis, and solar keratosis.
In preferred embodiments, the method of the invention is used to
inhibit growth, progression, and/or metastasis of cancers, in
particular those listed above.
Additional hyperproliferative diseases, disorders, and/or
conditions include, but are not limited to, progression, and/or
metastases of malignancies and related disorders such as leukemia
(including acute leukemias (e.g., acute lymphocytic leukemia, acute
myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid
tumors including, but not limited to, sarcomas and carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma.
Expression Vectors
Still other embodiments may concern DNA sequences comprising a
nucleic acid encoding an antibody, antibody fragment, cytokine or
constituent fusion protein of a bsAb, such as a DNL.TM. construct.
Fusion proteins may comprise an antibody or fragment or cytokine
attached to, for example, an AD or DDD moiety.
Various embodiments relate to expression vectors comprising the
coding DNA sequences. The vectors may contain sequences encoding
the light and heavy chain constant regions and the hinge region of
a human immunoglobulin to which may be attached chimeric, humanized
or human variable region sequences. The vectors may additionally
contain promoters that express the encoded protein(s) in a selected
host cell, enhancers and signal or leader sequences. Vectors that
are particularly useful are pdHL2 or GS. More preferably, the light
and heavy chain constant regions and hinge region may be from a
human EU myeloma immunoglobulin, where optionally at least one of
the amino acid in the allotype positions is changed to that found
in a different IgG1 allotype, and wherein optionally amino acid 253
of the heavy chain of EU based on the EU number system may be
replaced with alanine. See Edelman et al., Proc. Natl. Acad. Sci
USA 63: 78-85 (1969). In other embodiments, an IgG1 sequence may be
converted to an IgG4 sequence.
The skilled artisan will realize that methods of genetically
engineering expression constructs and insertion into host cells to
express engineered proteins are well known in the art and a matter
of routine experimentation. Host cells and methods of expression of
cloned antibodies or fragments have been described, for example, in
U.S. Pat. Nos. 7,531,327, 7,537,930, 7,785,880, 8,076,410,
8,153,433 and 8,372,603, the Examples section of each incorporated
herein by reference.
Kits
Various embodiments may concern kits containing components suitable
for treating or diagnosing diseased tissue in a patient. Exemplary
kits may contain one or more bsAbs, ADCs, interferons, and/or
checkpoint inhibitor antibodies as described herein. If the
composition containing components for administration is not
formulated for delivery via the alimentary canal, such as by oral
delivery, a device capable of delivering the kit components through
some other route may be included. One type of device, for
applications such as parenteral delivery, is a syringe that is used
to inject the composition into the body of a subject. Inhalation
devices may also be used. In certain embodiments, a therapeutic
agent may be provided in the form of a prefilled syringe or
autoinjection pen containing a sterile, liquid formulation or
lyophilized preparation.
The kit components may be packaged together or separated into two
or more containers. In some embodiments, the containers may be
vials that contain sterile, lyophilized formulations of a
composition that are suitable for reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or
dilution of other reagents. Other containers that may be used
include, but are not limited to, a pouch, tray, box, tube, or the
like. Kit components may be packaged and maintained sterilely
within the containers. Another component that can be included is
instructions to a person using a kit for its use.
EXAMPLES
The following examples are provided to illustrate, but not to
limit, the claims of the present invention.
Example 1
T-Cell Redirecting Bispecific Antibody DOCK-AND-LOCK.TM. (DNL.TM.)
Complexes
Several species of exemplary leukocyte redirecting bispecific
antibodies were made as DNL.TM. complexes, as described below. The
complexes were effective to induce an immune response against
appropriate target cells including, but not limited to,
Trop-2.sup.+ cancer cells.
Materials and Methods
General techniques for making and using DOCK-AND-LOCK.TM. (DNL.TM.)
complexes are described in the Examples below. An exemplary
leukocyte redirecting bispecific antibody with binding sites for
CD3 and CD19 was made as a DNL.TM. complex, referred to as (19)-3s
(FIG. 1). An anti-CD19 F(ab).sub.2 DNL module was constructed by
recombinant fusion of a dimerization and docking domain (DDD2) at
the carboxyl terminal end of the Fd chain. An anti-CD3-scFv module
was designed from Okt3 mAb with addition of an anchor domain (AD2)
and assembled in the format V.sub.H-L1-V.sub.K-L2-6H-L3-AD2 ("6H"
disclosed as SEQ ID NO:105), where the V domains were fused via a
flexible peptide linker and the AD2 peptide was preceded by a 6-His
linker (SEQ ID NO:105). The sequences of the anti-CD3 variable
regions, linkers and AD2 were as shown below.
TABLE-US-00017 VH sequence of anti-CD3 scFv (SEQ ID NO: 96)
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGY
INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY
DDHYSLDYWGQGTTLTVSS L1 Linker (SEQ ID NO: 97) GGGGSGGGGSGGGGS VK
sequence of anti-CD3 scFv (SEQ ID NO: 98)
DIVLTQSPAIIVISASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIY
DTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFG SGTKLEIKR L2
Linker (SEQ ID NO: 99) GGGGS Poly-His-L3 Linker (SEQ ID NO: 100)
HHHHHHGGGSG AD2 (SEQ ID NO: 101) CGQIEYLAKQIVDNAIQQAGC
Expression Vectors and DNL.TM. Modules--
DNL.TM. complexes were constructed comprising antibody moieties
against various disease-associated antigens, linked to an anti-CD3
antibody moiety, generally abbreviated as (X)-3s bsAbs. Independent
production cell lines were developed in SpESFX-10 mouse myeloma
cells (Rossi et al., 2011, Biotechnol Prog 27:766-75) for each of
the DNL.TM. modules used to make the (X)-3s bsAbs. A cDNA sequence
encoding the Okt3scFv-AD2 polypeptide (SEQ ID NOs:96-101) was
synthesized and cloned into the pdHL2 expression vector via 5' Xba
I and 3' Eag I restriction sites. The construct comprised the
V.sub.H domain fused to the V.sub.L in an scFv with the structure
V.sub.H-L1-V.sub.K-L2-6H-L3-AD2 ("6H" disclosed as SEQ ID NO:105).
The expressed protein had two amino acid substitutions from the
original Okt3 mAb. A cysteine residue in the CDR-H3 was changed to
serine (Kipryanov, 1997, J Immunol Methods 200:69-77). The
penultimate residue of the V.sub.L was changed from aspartate to
lysine.
The Okt3scFv-AD2 module was combined with various C.sub.H1-DDD2-Fab
modules to generate a panel of (X)-3s trivalent bsAbs (Table 6).
The C.sub.H1-DDD2-Fab-pdHL2 expression vectors were constructed as
described previously for similar constructs (Rossi et al., 2008,
Cancer Res 68:8384-92). Briefly, expression vectors encoding
C.sub.H1-DDD2-Fab were generated from the corresponding IgG-pdHL2
expression vectors by excising the coding sequence for the
C.sub.H1-Hinge-C.sub.H2-C.sub.H3 domains with Sac II and Eag I
restriction enzymes and replacing it with a 507 bp sequence
encoding C.sub.H1-DDD2, which was excised from the
C.sub.H1-DDD2-Fab-hA20-pdHL2 expression vector (Rossi et al., 2008,
Cancer Res 68:8384-92) with the same enzymes. C.sub.H1-DDD2-Fab
modules were derived from the humanized mAbs hA19 (anti-CD19),
labetuzumab (hMN-14, anti-CEACAM5), clivatuzumab (hPAM4,
anti-mucin), hMN-15 (anti-CEACAM6), hRS7 (anti-TROP-2), veltuzumab
(hA20, anti-CD20), hL243 (anti-HLA-DR) and epratuzumab (hLL2,
anti-CD22). The mAb designated hA19 was humanized from the mouse
anti-CD19 mAb B43 (Uckun et al., 1988, Blood 71:13-29). Each
expression vector was linearized by digestion with Sal I
restriction enzyme and used to transfect SpESFX-10 cells by
electroporation.
Clones were selected in media containing 0.2 .mu.M methotrexate
(MTX) and screened for protein expression by ELISA. Okt3scFv-AD2
was captured on Ni-NTA HisSorb plates (Qiagen) and detected with an
anti-AD2 mAb. C.sub.H1-DDD2-Fab modules were captured with
goat-anti-human-kappa chain and detected with
goat-anti-human-F(ab').sub.2-HRP. Productivity of
protein-expression was amplified by stepwise increases in MTX
concentration up to 3 .mu.M. Okt3scFv-AD2 and C.sub.H1-DDD2-Fab
modules were purified to homogeneity from the broth of roller
bottle cultures by affinity chromatography using Ni-SEPHAROSE.RTM.
and Kappa-Select resins, respectively. The DNL.TM. method was used
to assemble (X)-3s bsAbs via the site-specific conjugation of mole
equivalents of Okt3scFv-AD2 and C.sub.H1-DDD2-Fab modules. For
example, approximately 100 mg of (19)-3s were produced by combining
22 mg of Okt3scFv-AD2 with 80 mg of C.sub.H1-DDD2-Fab-hA19. The
mixture was reduced overnight at room temperature with 1 mM reduced
glutathione prior to the addition of 2 mM oxidized glutathione. The
(19)-3s was purified from the reaction mixture by sequential
affinity chromatography with Kappa-Select and Ni-SEPHAROSE.RTM..
Additional (X)-3s constructs were assembled at various scales
following a similar process.
TABLE-US-00018 TABLE 6 (X)-3s DNL .TM. Constructs Code Target
C.sub.H1-DDD2-Fab AD2-anti-CD3 (19)-3s CD19 C.sub.H1-DDD2-Fab-hA19
scFv-AD2-Okt3 (20)-3s CD20 C.sub.H1-DDD2-Fab-hA20 scFv-AD2-Okt3
(22)-3s CD22 C.sub.H1-DDD2-Fab-hLL2 scFv-AD2-Okt3 (C2)-3s HLA-DR
C.sub.H1-DDD2-Fab-hL243 scFv-AD2-Okt3 (M1)-3s MUC5AC
C.sub.H1-DDD2-Fab-hPAM4 scFv-AD2-Okt3 (14)-3s CEACAM5
C.sub.H1-DDD2-Fab-hMN-14 scFv-AD2-Okt3 (15)-3s CEACEAM6
C.sub.H1-DDD2-Fab-hMN-15 scFv-AD2-Okt3 (E1)-3s TROP-2
C.sub.H1-DDD2-Fab-hRS7 scFv-AD2-Okt3
Analytical Methods--
Size-exclusion high-performance liquid chromatography (SE-HPLC) was
performed with an Alliance HPLC System with a BIOSUITE.TM. 250,
4-.mu.m UHR SEC column (Waters Corp). Electrospray ionization time
of flight (ESI-TOF) liquid chromatography/mass spectrometry (LC-MS)
was performed with a 1200-series HPLC coupled with a 6210 TOF MS
(Agilent Technologies, Santa Clara, Calif.). The (19)-3s was
resolved by reversed phase HPLC (RP-HPLC) at 60.degree. C., using a
14-min gradient of 30-80% acetonitrile in 0.1% aqueous formic acid
with an Aeris widepore 3.6 .mu.m C4 column (Phenomenex). For the
TOF MS, the capillary and fragmentor voltages were set to 5500 and
300 V, respectively.
Cell Lines and Reagents--
Raji, Ramos, Daudi, LS174T and Capan-1 cell lines were purchased
from the American Type Cell Culture Collection (ATCC, Manassas,
Md.) and Nalm-6 cells were purchased from Deutsche Sammlung von
Mikroorganismen and Zellinien (DSMZ, Braunchweig, Germany). All
cell lines, except Capan-1, were maintained in RPMI-1640 containing
10% FBS, 1% L-glutamine, 1% penicillin-streptomycin and 1% MEM
nonessential amino acids. Capan-1 cells were maintained with 20%
FBS. All cell culture media and supplements were purchased from
Life Technologies (Carlsbad, Calif.).
PBMCs and T Cell Isolation--
Human peripheral blood mononuclear cells (PBMC) were purified from
whole donor blood (Blood Center of NJ, East Orange, N.J.) using
UNI-SEP.sub.MAXI tubes (Novamed, Ltd, Jerusalem, Israel).
CD3-positive T cells were isolated from PBMCs by negative selection
using the Pan T Cell Isolation Kit (Miltenyi Biotec, Auburn,
Calif.), according to the manufacturer's protocol. Efficiency of T
cell isolation was assessed by FACS after staining the enriched T
cells with anti-CD3-PE antibody. In some cases, further staining
with CD-19 and CD-14 was performed as well to identify
contaminating cells.
T Cell Activation--
Isolated T cells were plated in 6-well tissue culture plates at a
final density of 2.25.times.10.sup.6 cells/well. Daudi cells were
added to some wells at a final density of 1.5.times.10.sup.6
cells/well, other wells were left to contain only T cells.
Alternatively, PBMCs were added to 6-well tissue culture plates at
a final cell density of 6.times.10.sup.6 cells/well. The volume of
each well was brought up to 3 mL. To the appropriate wells, 3 ng/mL
of (19)-3s, (M1)-3s or (19)-DDD2 was added. After incubation
overnight at 37.degree. C., 1 mL of each sample was removed. The
cells were pelleted and labeled on ice with CD69-APC and CD3-PE for
20 minutes. Cells were washed 2 times with 1% BSA in PBS and
analyzed using a FACSCALIBER.TM. flow cytometer (BD Biosciences,
San Jose, Calif.).
T-Cell Proliferation--
PBMCs were seeded in T25 flasks at a concentration of
1.times.10.sup.6 cells/mL containing the specified reagents. For B
cell-depleted flasks, B cells were removed by negative selection
using a B-cell isolation kit from Miltenyi according to
manufacturer's protocol. On select days, 100 .mu.L of media was
removed from each flask, labeled with anti-CD7-APC for 20 minutes
on ice, washed once and resuspended in 300 .mu.L of 1% BSA/PBS
containing 7-AAD. For each sample, the entire volume is analyzed
using a FACSCALIBER.TM. flow cytometer. Each sample is counted in
duplicate. Analysis is performed using FlowJo Software. For each
sample, dead (7-AAD+) cells, and debris (based on forward vs. side
scatter) was removed. Finally, live CD7+ cells were selected and
plotted using Prism software.
Cell Binding Assays (Jurkat/Capan-1)--
Jurkat cells were stained with PKH26 Red Fluorescent Cell Linker
Kit (Sigma) according to manufacturer's protocol. Capan-1 cells
were stained with 5 .mu.M CFSE (carboxyfluorescein diacetate
succinimidyl ester, Life Technologies) according to manufacturer's
protocol. Labeled Capan-1 cells were added to 8-well chamber slides
(ThermoWaltham, Mass.) and allowed to attach overnight. The
following day, media was removed and PKH26-labeled Jurkat cells
were added in media containing 0.1 .mu.g/mL of (E1)-3s, (M1)-3s or
(19)-3s. Following a 1-hour incubation at 37.degree. C., slides
were washed with PBS to remove any unbound cells and observed by
fluorescence microscopy.
Cell Binding Assays (Jurkat/Daudi)--
Jurkat and Daudi cells were labeled with anti-CD3-PE and
anti-CD20-FITC, respectively. Labeled cells were then coincubated
at a 2.5:1 ratio with 0.1 .mu.g/mL (19)-3s for 30 minutes at room
temperature. Aliquots of cells were then observed by fluorescence
microscopy.
Cytotoxicity Assay (Hematologic Tumor Cell Lines)--
Target cells were labeled with PKH67 Green Fluorescent Cell Linker
Kit (Sigma) according to the manufacturer's protocol. Briefly,
5.times.10.sup.6 target cells were resuspended in 250 .mu.L of
diluent C. In a second tube 1 .mu.L of PKH26 dye is added to 250
.mu.L of diluent C. The cell suspension is then added to the dye
solution, mixed thoroughly and incubated at RT for 2 minutes. The
reaction was quenched by adding an equal volume of FBS. The labeled
cells were then washed 3 times with complete RPMI. Unstimulated,
isolated T cells were used as effector cells. Effector cells and
PKH67-labeled target cells were combined at a 10:1 ratio and plated
in 48-well plates containing serial dilutions of (19)-3s or
(14)-3s. Each well contained 5.times.10.sup.4 target cells and
5.times.10.sup.5 effector cells. Jeko-1 assays were performed in
20% RPMI. Plates were incubated for 18-24 hours in a 37.degree. C.
incubator containing 5% CO.sub.2. Following incubation, all cells
were removed from 48-well plates into flow cytometer tubes and
resuspended in 1% BSA/PBS containing 1 ug/mL of 7AAD, to
distinguish live from dead cells, and 30,000 COUNTBRIGHT.TM.
Absolute Counting Beads (Life Technologies). Cells were analyzed on
a FACSCALIBER.TM. flow cytometer. For each sample, 8,000
COUNTBRIGHT.TM. beads were counted as a normalized reference. Data
were analyzed using FlowJo software (Treestar, Inc., Ashland,
Oreg.). For each sample, dead cells and debris were excluded and
total live target cells were counted.
Cytotoxicity Assay (Solid Tumor Cell Lines)--
Target cells were labeled with PKH67 Green Fluorescent Cell Linker
Kit (Sigma) following the same procedure as for staining with
PKH23. Effector cells used were as follows: For Capan-1 assays,
CD8+ enriched T cells were used, following purification from a CD8+
enrichment column (R&D Systems, Minneapolis, Minn.). For LS174T
cells: Stimulated T cells were used after incubation of PBMC for 5
days in media containing 25 U/mL IL-2 and 50 ng/mL Okt3 Mab,
followed by 2 days incubation in media containing 25 U/mL IL-2
alone. Effector cells and PKH67-labeled target cells were combined
at a 3:1 ratio (5.times.10.sup.4 target cells and
1.5.times.10.sup.5 effector cells/well) and plated over 48-well
plates containing serial dilutions of (E1)-3s, (14)-3s or (19)-3s.
Capan-1 assays were performed in 20% RPMI. Plates were incubated
for 42-48 hours in a 37.degree. C. incubator containing 5%
CO.sub.2. Following incubation, suspension cells were combined with
trypsinized attached cells from all wells and transferred into flow
cytometer tubes. Cells were washed one time and resuspended in 1%
BSA/PBS containing 1 ug/mL of 7AAD, to distinguish live from dead
cells, and 30,000 COUNTBRIGHT.TM. Absolute Counting Beads. Cells
were analyzed on a FACSCALIBER.TM. flow cytometer. For each sample,
8,000 COUNTBRIGHT.TM. beads were counted as a normalized reference.
Data were analyzed using FlowJo software (Treestar, Inc., Ashland,
Oreg.). For each sample, dead cells and debris were excluded and
total live target cells were counted.
In Vivo Efficacy--
Female NOD/SCID mice, 8 weeks old, were purchased from Charles
River (Wilmington, Mass.). Mice were injected s.c. with a mixture
of Raji (1.times.10.sup.6) and human PBMCs (5.times.10.sup.6 cells)
mixed 1:1 with matrigel. Therapy began 1 hour later. Treatment
regimens, dosages, and number of animals in each experiment are
described in the Results. Animals were monitored daily for signs of
tumor out-growth. Once tumors appeared, they were measured twice
weekly. Tumor volume (TV) was determined by measurements in two
dimensions using calipers, with volumes defined as:
L.times.w.sup.2/2, where L is the longest dimension of the tumor
and w the shortest. Efficacy was determined by a log-rank test
using Prism GraphPad software (v5; LaJolla, Calif.) on Kaplan-Meier
curves using survival surrogate endpoints as time for tumor
progression (TTP) to 1.0 cm.sup.3. Significance was considered at
P<0.05.
Results
Construction and Biochemical Analysis of Leukocyte Redirecting
Bispecific Antibodies.
The DNL.TM. method was used to generate a panel of (X)-3s,
leukocyte redirecting bsAbs for targeting of various
tumor-associated antigens including CD19, CD20, HLA-DR, TROP-2,
CEACAM5 and MUC5AC. The purity of these structures was demonstrated
by SE-HPLC and SDS-PAGE analysis, where only bands representing the
three constituent polypeptides (Okt3scFv-AD2, hA19-Fd-DDD2 and hA19
kappa) were evident (data not shown). LC-MS analysis identified a
single RP-HPLC peak having a deconvoluted mass spectrum consistent
(mass accuracy=11 ppm) with the calculated mass (137432.37 Da) of
(19)-3s from its deduced amino acid sequence, including the
predicted amino-terminal pyroglutamates on the Okt3scFv-AD2 and
each of the two C.sub.H1-DDD2-hA19 Fd chains (data not shown). No
additional post-translational modifications, including
glycosylation were indicated.
Immune Synapse Formation Between Daudi Burkitt Lymphoma and T
Cells, Mediated by (19)-3s.
The effects of the leukocyte redirecting (19)-3s DNL.TM. complex on
targeting effector T cells to CD19.sup.+ lymphoma cells was
examined (FIG. 2). Freshly isolated T cells were combined with
Daudi cells at an E:T ratio of 2.5:1. Cells were treated with 0, 1
or 5 .mu.g/mL of (19)-3s DNL.TM. complex for 30 min at room
temperature prior to analysis by flow cytometry. Anti-CD20-FITC and
anti-CD7-APC were used to identify Daudi and T cells, respectively.
Co-binding was indicated as the % of CD20.sup.+/CD7.sup.+ events.
After treatment with (19)-3s, 45.5% of flow events were CD20/CD7
dual-positive, indicating synapsed Daudi and T cells (FIG. 2A),
compared to 2% measured for the mixed cells without antibody (FIG.
2B). Addition of (19)-3s resulted in association of >90% of the
Daudi with T cells (FIG. 2C). These results show that the (19)-3s
DNL.TM. complex was effective to direct T cells to the targeted
antigen-expressing lymphoma cells.
Synapse formation between T cells and target lymphoma cells was
demonstrated by fluorescence microscopy (FIG. 3) Jurkat (T cells)
and Daudi (B cells) were combined at a 1:1 ratio, treated with 0.1
.mu.g/mL of the (19)-3s DNL.TM. complex for 30 minutes and stained
with anti-CD20-FITC (FIG. 3A) and anti-CD3-PE (FIG. 3B), prior to
analysis by fluorescence microscopy. The merged image (FIG. 3C)
reveals synapse formation between green-stained Daudi and
red-stained Jurkat cells. Synapse formation was not evident in the
absence of (19)-3s (FIG. 3D). FIG. 3C demonstrates that the target
lymphoma cells are in direct contact with the targeted T cells.
A dose-response series was performed for (19)-3s mediated
association of T cells to an exemplary B-cell lymphoma line (FIG.
4). As shown in FIG. 4, under the conditions of this experiment,
saturation of (19)-3s-mediated cell-to-cell association of T cells
to target cells was reached at a concentration between 0.037 and
0.111 .mu.g/ml of the DNL.TM. complex.
FIG. 5 shows a comparision of the relative efficacies of BITE.RTM.
(FIG. 5A), DART.TM. (FIG. 5A) and DNL.TM. (FIG. 5B)
anti-CD3.times.anti-CD19 complexes for redirecting T cells to
targeted CD19.sup.+ B cells. The data for BITE.RTM. and DART.TM.
was obtained from Moore et al. (2011, Blood 117:4542-51). At the
lowest concentration tested of 0.0005 .mu.g/ml, the (19)-3s DNL.TM.
complex was more effective than BITE.RTM. or DART.TM. at targeting
T cells to B-cell lymphoma (FIG. 5). The (19)-3s DNL.TM. complex
also induced a slightly higher maximum level of cell-to-cell
association than the comparable BITE.RTM. and DART.TM. complexes
(FIG. 5A). Although difficult to extrapolate from the single data
points generated for the (19)-3s DNL.TM. complex, the EC.sub.50
levels appeared to be similar for BITE.RTM., DART.TM. and DNL.TM.
(FIG. 5).
(19)-3s, (E1)-3s and (M1)-3s-Mediated Cell-Cell Association of T
Cells to Target Tumor Cells.
To evaluate the ability of the T-cell redirecting BsAbs to
facilitate the association of T cells to their target tumor cells,
Jurkat T cells were coincubated with target tumor cells containing
(X)-3s and evaluated by flow cytometry and fluorescence microscopy.
Jurkat T cells are a CD4+ T cell leukemia line, chosen for their
ability to demonstrate T cell binding without depletion of the FITC
labeled Daudi cells in the presence of various concentrations of
(19)-3s and analyzed by flow cytometry for the detection of double
positive (CD3+CD20+) populations indicating T cell-B cell
associated complexes. An apparent cell-cell association was seen
following treatment with 0.5 ng/mL of (19)-3s and after treatment
with 0.1 .mu.g/mL over 25% of the cell population existed in a
cell-cell association (FIG. 5). Fluorescent microscopy supports
this data, as immune synapses are evident following treatment with
0.1 .mu.g/mL (19)-3s (FIG. 4). No synapse formation was seen in the
absence of (19)-3s (data not shown).
This cell-cell association was observed in the pancreatic tumor
line Capan-1 as well (FIG. 6). Capan-1 expresses high levels of
TROP2 and moderate levels of MUC5AC. Therefore, both the
TROP2-targeting bsAb, (E1)-3s (FIG. 6C), and MUC5AC-targeting bsAb,
(M1)-3s (FIG. 6B) were compared to the non-targeting control bsAb,
(19)-3s (FIG. 6A). CF SE-labeled Capan-1 cells were coincubated
with PKH26-labeled Jurkat in the presence of these bsAbs.
Fluorescent microscopy revealed, as expected, large T-cell/Capan
complexes mediated by (E1)-3s, followed by smaller, yet substantial
complexes mediated by (M1)-3s and relatively low complex formation
following (19)-3s treatment (FIG. 6).
(19)-3s Specifically Induces T Cell Activation and
Proliferation.
The ability of (19)-3s to activate T cells was evaluated either in
PBMCs (FIG. 7A), or T cells coincubated with Daudi B cells (FIG.
7B), by measuring the expression levels of CD69, an early marker of
T cell activation. Treatment with 3 ng/mL of (19)-3s induced T cell
activation in T cells coincubated with Daudi B cells as indicated
by a >50-fold increase in CD69 expression compared with
non-targeting control antibodies, (19)-DDD2 and (M1)-3s, as well as
T cells treated with (19)-3s without Daudi target cells (FIG. 7B).
Similar results were observed when the antibodies were incubated
with PBMCs, containing both T and B cells; (19)-3s stimulated CD69
expression levels >20-fold higher than non-targeting controls
(FIG. 7A). In the absence of target cells, purified T cells treated
with (19)-3s did not show activation (FIG. 7C).
T cell proliferation, as another indication of T cell activation,
was evaluated after treatment of PBMCs with various CD3-targeting
antibodies. (19)-3s at 3 nM or 30 pM induced T cell proliferation
similar to that of the positive control IL-2/PHA (FIG. 8A).
Non-targeting control antibody, (14)-3s, shows some non-specific T
cell proliferation at the highest (3 nM) concentration (FIG. 8A).
However, T cell proliferation was not observed in PBMCs depleted of
B cells (FIG. 8B), suggesting that target cells are necessary for
specific (19)-3s induced T cell proliferation.
(X)-3s Re-Directed T-Cell Mediated Killing of Malignant Cell
Lines.
The cytotoxicity of each leukocyte targeting molecule was evaluated
by its ability to mediate lysis of specific tumor target cells. For
the hematologic tumor cell lines, a 10:1 E:T ratio using an
unstimulated, enriched T cell population as the effector cells in
an 18-24 hour assay demonstrated the optimal assay conditions. The
CD19-targeting bsAb, (19)-3s induced the most potent specific
killing of the relatively low CD19-expressing cell lines Ramos
(IC.sub.50=0.17 pM, Lysis.sub.Max=79%) Daudi (IC.sub.50=1 pM,
Lysis.sub.Max=60%), and Nalm6 (IC.sub.50=6 pM, Lysis.sub.Max=93%)
(FIG. 9A). Interestingly, the high CD19-expressing cell lines,
Namalwa (IC.sub.50=63 pM, Lysis.sub.Max=60%) and Raji (IC.sub.50=3
nM, Lysis.sub.Max=41%) were the least sensitive to (19)-3s (FIG.
9A). The non-targeting (14)-3s DNL.TM. construct had little
cytotoxic effect in any of the cell lines tested (FIG. 9B).
Consistent cytotoxic effects of the (19)-3s construct on the Nalm-6
ALL cell line were obtained with PBMCs obtained from two different
donors (FIG. 9C).
The in vitro cytotoxic effects of (20)-3s, (22)-3s and (C2)-3s
T-cell redirecting bsAbs were determined in several cell lines
(FIG. 10). The CD22-targeting bsAb, (22)-3s, demonstrated potent
(IC.sub.50=5 pM, Lysis.sub.Max=60%) specific T-cell mediated lysis
in the CD22-positive Daudi cell line (FIG. 10C), but not in the
CD22-negative Namalwa cells (FIG. 10A).
The CD20-targeting bsAb, (20)-3s demonstrated the highest potency
in the higher-expressing CD20 cell lines, Daudi (IC.sub.50=<0.3
pM, Lysis.sub.Max=90%) (FIG. 10C) and Jeko (IC.sub.50=1 pM,
Lysis.sub.Max=90%) (FIG. 10B), compared to the lower
CD20-expressing Namalwa cell line (IC.sub.50=30 pM,
Lysis.sub.Max=53%) (FIG. 10A).
The HLA-DR-targeting bsAb, (C2)-3s was tested in the HLA-DR
expressing Jeko-1 cell line (IC.sub.50=20 pM, Lysis.sub.Max=88%)
(FIG. 10B).
At an E:T ratio of 10:1, using isolated T cells as effector cells,
the bsAbs induced potent T cell-mediated cytotoxicity in various B
cell malignancies, including Burkitt lymphoma (Daudi, Ramos,
Namalwa) mantle cell lymphoma (Jeko-1) and acute lymphoblastic
leukemia (Nalm-6) (Table 7). A non-tumor binding control, (14)-3s,
induced only moderate T-cell killing at >10 nM. The nature of
the antigen/epitope, particularly its size and proximity to the
cell surface, appears to be more important than antigen density for
T-cell retargeting potency (Table 7). It is likely that (20)-3s is
consistently more potent than (19)-3s and (C2)-3s, even when the
expression of CD19 or HLA-DR is considerably higher than CD20, as
seen with Namalwa and Jeko-1, respectively (Table 7). This is
likely because the CD20 epitope comprises a small extracellular
loop having close proximity to the cell surface. When compared
directly using Daudi, (22)-3s was the least potent. Compared to
CD19 and CD20, CD22 is expressed at the lowest density, is a
rapidly internalizing antigen, and its epitope is further away from
the cell surface. Each of these factors may contribute to its
reduced potency. Finally, sensitivity to T-cell retargeted killing
is cell-line-dependent, as observed using (19)-3s, where Raji
(IC.sub.50>3 nM) is largely unresponsive yet Ramos (IC.sub.50=2
pM) is highly sensitive, even though the former expresses higher
CD19 antigen density (Table 7).
In conclusion, (19)-3s, (20)-3s, (22)-3s and (C2)-3s bind to T
cells and target B cells simultaneously and induce T-cell-mediated
killing in vitro. The modular nature of the DNL method allowed the
rapid production of several related conjugates for redirected
leukocyte killing of various B cell malignancies, without the need
for additional recombinant engineering and protein production. The
close proximity of the CD20 extracellular epitope to the cell
surface resulted in the highest potency for (20)-3s.
TABLE-US-00019 TABLE 7 Ex vivo re-directed T-cell killing Antigen
Expression.sup.2 IC.sub.50.sup.4 (pM) Cell Line Type.sup.1 CD19
CD20 CD22 HLA- (19)-3s (20)- (22)-3s (C2)- Daudi BL 1.00 1.00 1.00
1.00 1 0.3 6 N.D. Ramos BL 0.76 0.65 0.26 0.36 2 0.4 N.D. 2 Nalm-6
ALL 1.63 0.05 0.19 0.17 6 N.D. N.D. N.D. Namalwa BL 0.76 0.11 0.05
0.40 63 30 >3000 N.D. Raji BL 1.41 0.69 0.59 0.84 >3000 N.D.
N.D. N.D. Jeko-1 MCL 0.89 1.02 0.05 1.06 3000 1 N.D. 20 .sup.1BL,
Burkitt lymphoma; ALL, acute lymphoblastic leukemia; MCL, mantle
cell lymphoma. .sup.2Expression level determined by flow cytometry
and normalized to that of Daudi. .sup.3IC.sub.50, the picomolar
concentration that achieved 50% target cell killing.
The in vitro cytotoxic effects of leukocyte redirecting bsAbs were
also determined in solid tumor cells (FIG. 11). For the solid tumor
cell lines, optimal assay conditions were determined to be a 3:1
E:T ratio using stimulated T cells in a 42-48 hour assay. Each bsAb
induced specific T-cell mediated lysis of the tumor target cells.
The CEACAM5-expressing human colon adenocarcinoma cell line,
LS-174T, demonstrated potent specific lysis (IC.sub.50=2 pM)
following treatment with (14)-3s (FIG. 11A). (E1)-3s mediated
potent specific lysis of the TROP2 expressing Capan-1 human
pancreatic adenocarcinoma cell line (IC.sub.50=29 pM) (FIG. 11B).
The gastric carcinoma cell line NCI-N87, which expresses high
levels of both CEACAM6 and TROP 2 demonstrated very potent specific
lysis to both T-cell targeting molecules, (15)-3s and (E1)-3s
(IC.sub.50=3 pM and 0.85 pM respectively) (FIG. 11C). The
non-targeting control antibody, (19)-3s, induced low (<20%)
non-specific lysis at concentrations >1 nM for Capan-1 and
LS174T, and moderate (.about.40%) non-specific lysis in NCI-N87
cells (FIG. 11A-C). A summary of the in vitro cytotoxicity data for
various leukocyte redirecting bsAbs in a variety of tumor cell
lines is shown in FIG. 12. The various constructs showed a maximal
cell lysis of up to 90% or more of the targeted tumor cells, with
IC.sub.50 values for cell lines expressing the targeted antiben
that were generally in the low picomolar range (FIG. 12).
Example 2
In Vivo Studies of Leukocyte Redirecting DNL.TM. Complex
One potential limitation of small (<60 kDa) scFv-based
constructs, such as BITE.RTM. and DART.TM., is the requirement for
administration by long-term continuous infusion, due to their
toxicity and rapid clearance from circulation. Because the
molecular size of DNL.TM. bsAbs is above the threshold typically
associated with renal clearance, it should exhibit slower clearance
from circulation. We measured the pharmacokinetic parameters in
mice following a single bolus i.v. injection of 5 mg/kg of the
(19)-3s bsAb (data not shown). A biphasic clearance was observed
with a t1/2.alpha. and t1/2.beta. of 1.1 and 5.1 h, respectively,
resulting in an area under the curve of 1880 pmol*h/mL (data not
shown), which was nearly 6-fold greater than that reported for
MT103 (anti-CD19.times.anti-CD3 BITE.RTM.) administered at the same
molar concentration (US Patent US2010/0303827A1). The major
difference is apparently a longer t1/2.alpha. for (19)-3s (data not
shown). Because of the potentially advantageous properties of
(19)-3s, we evaluated the possibility of using less frequent dosing
schedules rather than daily dosing, which is typically used for
BITE.RTM. in animal studies.
A pilot study was performed using Raji human Burkitt lymphoma
xenografts in NOD/SCID mice reconstituted with human PBMCs (FIG.
13, FIG. 14). Raji cells (1.times.10.sup.6 cells/mouse) were
combined with freshly isolated PBMCs (5.times.10.sup.6 cells/mouse)
from a single healthy donor, mixed 1:1 with matrigel, and injected
SC into all of the animals in the study on Day 0. Groups of 5 mice
received i.v. injections of (19)-3s totaling 130 .mu.g as a single
dose on Day 0 (FIG. 13B), three doses of 43 .mu.g (Days 0, 2 and 4)
(FIG. 13C) or five daily doses of 26 .mu.g (Days 0-5) (FIG. 13D).
The untreated group (FIG. 13A), which was inoculated with the same
cell mixture but did not receive (19)-3s, had a median survival
time (MST) of 31 days. Each therapy regimen improved survival
(P.ltoreq.0.05), with the three dose (every other day) schedule
providing the greatest survival benefit (MST=91 days; P=0.0018 by
log-rank analysis).
A follow-up study was begun to determine the efficacy of less
frequent dosing (FIG. 14). Groups of 9 NOD/SCID mice were
inoculated with Raji and PBMCs in a similar fashion as above. In
this study, therapy was extended to two weeks, compared to one week
in the first study. Groups received i.v. injections of (19)-3s
totaling 360 .mu.g as 2.times.130-.mu.g (FIG. 14B),
4.times.65-.mu.g (FIG. 14D) or 6.times.43-.mu.g doses over two
weeks (FIG. 14E). An additional group was administered
2.times.130-.mu.g doses SC, instead of i.v. (FIG. 14C). For
comparison, control groups of untreated mice (FIG. 14A) or mice
treated with non-targeting (M1)-3s antibody (FIG. 14F) were
prepared. As of Day 28, each of the (19)-3s treatment groups had
significantly smaller AUC than the untreated control (P<0.05).
Surprisingly, two weekly doses via the SC route was apparently as
effective as greater frequency i.v. dosing.
In vivo studies were also performed using solid tumors (FIG. 15).
NOD/SCID mouse xenografts were prepared as described above, for the
LS174T colon adenocarcinoma (FIG. 15A, FIG. 15B) or Capan-1
pancreatic carcinoma (FIG. 15C, FIG. 15D). In each case, mice
administered the targeting (E1)-3s (FIG. 15B) or (14)-3s (FIG. 15D)
bsAb DNL.TM. constructs showed improved survival compared to
controls.
In conclusion, the leukocyte-retargeting bsAbs, including (19)-3s,
(E1)-3s and (M1)-3s DNL.TM. constructs, mediated synapse formation
between T cells and B cells, colon adenocarcinoma or pancreatic
carcinoma cells, respectively, via monovalent and bivalent binding
to CD3 and CD19, respectively. T-cell activation, proliferation and
target cell killing were induced by the DNL.TM. bsAbs at pM
concentrations in an ex vivo setting. Advantageous properties of
the DNL.TM. bsAbs, including bivalent tumor binding and slower
clearance, would allow for less frequent dosing and possibly SC
administration, compared to BITE.RTM. or DART.TM. constructs, which
are administered i.v. daily in animal models and as a continuous
infusion in the clinic. The modular nature of the DNL.TM. method
allows the rapid production of a large number of related conjugates
for redirected leukocyte killing of various malignancies, without
the need for additional recombinant engineering and protein
production.
The person of ordinary skill in the art will realize that other
antibodies that bind to CD3 or other leukocyte antigens, as well as
other antibodies that bind to Trop-2 or other disease-associated
antigens are known in the art and any such antibody can be used to
make F(ab).sub.2, scFv or other antibody fragments using techniques
well known in the art. Such alternative antibodies or fragments
thereof may be utilized in the instant methods and compositions. As
discussed below, methods of making DOCK-AND-LOCK.TM. (DNL.TM.)
complexes may be applied to incorporate any known antibodies or
antibody fragments into a stable, physiologically active
complex.
Example 3
Interferon-.alpha. Enhances the Cytotoxic Effect of
Anti-Trop-2.times.Anti-CD3 Bispecific Antibodies
The therapeutic efficacy of an anti-human Trop-2.times.anti-human
CD3 bispecific antibody ((E1)-3s), made from hRS7 and OKT3 as a
DNL.TM. complex, was tested for its ability to delay tumor
outgrowth of Capan-1 human pancreatic adenocarcinoma tumor cells
when mixed with human T-cells and injected into mice. The effect of
interferon-.alpha.(either in the form of E1*-2b or PEGASYS.RTM.)
when combined with this therapy was also evaluated.
Methods
Five week-old female NOD/SCID mice were injected s.c. with a
mixture of Capan-1 (5.times.10.sup.6) and human T-cells
(2.5.times.10.sup.6 cells) mixed 1:1 with matrigel (E:T ratio of
1:2). There were six different treatment groups of 8 mice each.
Treatment consisted of one group receiving 47 .mu.g (E1)-3s i.v.
every day for five days starting 1 hour after the administration of
the Capan-1/T-cell mixture. Two groups were treated with equimolar
amounts of IFN, one received the DNL molecule made from
IFN-.alpha.2b-DDD2-CK-hRS7 IgG1 (E1*-2b; 2.5 .mu.g s.c.
weekly.times.4 wks) while another received PEGASYS.RTM. (Roche; 0.6
.mu.g s.c. weekly.times.4 wks). Two other groups received a
combination of (E1)-3s plus E1*2b or (E1)-3s plus PEGASYS.RTM.. The
final group control group remained untreated. Table 8 summarizes
the various treatment groups.
TABLE-US-00020 TABLE 8 Treatment Groups for (E1)-3s Therapy (E1)-3s
Therapy of a Human Pancreatic Carcinoma Xenograft (Capan-1) in
NOD/SCID Mice Group (N) Amount Injected Schedule 1 8 Untreated N.A.
2 8 (E1)-3s qdx5 (47 .mu.g i.v.) 3 8 E1*-2b qwkx4 (2.5 .mu.g s.c.)
4 8 PEGASYS .RTM. qwkx4 (0.6 .mu.g s.c.) 5 8 (E1)-3s + qdx5 +
E1*-2b qwkx4 6 8 (E1)-3s + qdx5 + PEGASYS qwkx4
Mice were monitored daily for signs of tumor out-growth. All
animals had their tumors measured twice weekly once tumors began to
come up. Mice were euthanized for disease progression if their
tumor volumes exceeded 1.0 cm.sup.3 in size.
Results
Mean tumor volumes for the various groups are shown in FIG. 16. The
data containing PEGASYS.RTM. groups (FIG. 16B) are shown on a
separate graph from the E1*2b groups (FIG. 16A) for clarity. All
treatments were significantly better at controlling tumor growth in
terms of area-under-the-curve (AUC) when compared to the untreated
mice out to day 29, which was when the first mouse in the untreated
group was euthanized for disease progression (P<0.0009;
AUC.sub.29 days). Combining (E1)-3s with PEGASYS.RTM. resulted in
the best anti-tumor response overall in terms of tumor out-growth
(FIG. 16B). This treatment was significantly better than any of the
individual treatments (P<0.042; AUC) as well as superior to the
combination of (E1)-3s plus E1*-2b (P=0.0312; AUC.sub.53 days)
(FIG. 16A). The combination of (E1)-3s plus E1*2b could
significantly control tumor growth when compared to E1*2b or
PEGASYS.RTM. alone (P<0.0073; AUC.sub.46 days) but not (E1)-3s
alone (FIG. 16A-B). There were no significant differences between
mice treated with (E1)-3s, PEGASYS.RTM., or E1*-2b (FIG.
16A-B).
In terms of survival, all treatments provide a significant survival
benefit when compared to the untreated mice (P<0.0112; log-rank)
(FIG. 17). As of day 81, there was no significant difference in
median survival times (MST) between mice treated with the
combination of (E1)-3s plus E1*-2b and those treated (E1)-3s plus
PEGASYS.RTM. (MST=79.5 and >81 days, respectively) (FIG. 17).
The mice treated with (E1)-3s plus PEGASYS.RTM. had a significantly
improved survival outcome than any of the individual treatments
(P<0.0237) (FIG. 17). Mice treated with (E1)-3s plus E1*2b had a
survival benefit when compared to mice treated with E1*-2b alone
(MST=53 days; P<0.0311) but not when compared to mice treated
with just (E1)-3s or PEGASYS.RTM. alone (MST=68 and 53 days,
respectively) (FIG. 17). Treatment with (E1)-3s provided a
significant improvement in survival when compared to mice treated
with E1*-2b (P=0.0406) but not when compared to mice treated with
PEGASYS.RTM. alone (FIG. 17). There was no significant differences
between mice treated with only E1*2b and those treated with
PEGASYS.RTM. alone (FIG. 17).
The results demonstrate that addition of interferon-.alpha.
provides a substantial increase in survival and decrease in tumor
growth when combined with a leukocyte redirecting bsAb. The person
of ordinary skill will realize that the improved efficacy observed
with addition of type I or type III interferons
(interferon-.alpha., interferon-.beta., or interferon-.lamda.) is
not limited to the specific (E1)-3s bsAb, but will be observed with
other leukocyte redirecting bsAbs, made either as DNL.TM. complexes
or in other forms, such as BITE.RTM. or DART.TM..
Example 4
Further Studies on Interferon-.alpha. Combination Therapy with
Leukocyte-Redirecting Bispecific Antibodies
In the Example above, the combination of (E1)-3s plus PEGASYS.RTM.
proved to be a very effective treatment in the control of tumor
growth. In order to confirm these results and extend them, a study
was performed in which two new groups were added. First, a control
group for (E1)-3s was included in which an equimolar amount of TF12
was administered to animals. TF12 consists of two hRS7-Fab
molecules linked to one non-targeting 679 Fab (anti-HSG).
Additionally, since Capan-1 is sensitive to IFN, another group was
added in which the effect of PEGASYS.RTM. on Capan-1 tumor growth
was assessed without the benefit of T cells.
After the mice (40) were injected with the Capan-1/T-cell mixture,
they were randomized into five treatment groups. One hour later,
one group of 11 mice received 47 .mu.g (E1)-3s i.v. every day
starting 1 h post-tumor cell injection and continued for four more
consecutive days (qd.times.5). One group of 7 animals received
interferon in the form of PEGASYS.RTM. s.c. on a weekly basis for
four weeks. Another group received a combination of (E1)-3s i.v.
plus PEGASYS.RTM. s.c. Untreated control animals receive Capan-1/T
cells but no treatment. A further control group received TF12 at
amounts equivalent to the (E1)-3s in terms of moles (57 .mu.g
qd.times.5). Group 6 mice (8 animals) received a separate injection
of only Capan-1 cells (i.e., no T cells) and was treated with
PEGASYS.RTM.. All therapy injections were in a volume of 100 .mu.L.
Table 9 summarizes the various groups
TABLE-US-00021 TABLE 9 Treatment Groups for (E1)-3s and TF12
Therapy (E1)-3s Therapy of a Human Pancreatic Carcinoma Xenograft
(Capan-1) in NOD/SCID Mice Group (N) Amount Injected Schedule 1 7
Untreated N.A. (Capan-1 + T cells only) 2 11 (E1)-3s qdx5 (47 .mu.g
i.v.) 3 7 TF12 qdx5 (57 .mu.g i.v.) 4 7 PEGASYS .RTM. qwkx4 (0.6
.mu.g s.c.) 5 8 (E1)-3s + qdx5 + PEGASYS .RTM. qwkx4 6 8 PEGASYS
.RTM. qwkx4 (0.6 .mu.g s.c.) (Capan-1 cells only)
Mice were monitored daily for signs of tumor out-growth. All
animals had their tumors measured twice weekly once tumors began to
come up. Mice were euthanized for disease progression if their
tumor volumes exceeded 1.0 cm.sup.3 in size.
Results
Mean tumor growth (FIG. 18) and survival curves (FIG. 19) are
shown. While not different from each other, mice treated with
(E1)-3s, PEGASYS.RTM., or PEGASYS.RTM. (without T cells),
demonstrated significant anti-tumor effects when compared to TF12
and untreated control groups (P<0.0102; AUC). On the day this
experiment ended (day 59), the mean tumor volume for the mice
treated with the combination of (E1)-3s plus PEGASYS.RTM. was
0.083.+-.0.048 cm.sup.3. Overall, this treatment group demonstrated
a significant anti-tumor effect when compared to all the other
treatment groups (P<0.0072; AUC).
Each individual treatment (PEGASYS.RTM., PEGASYS.RTM. without T
cells, and (E1)-3s) significantly improved survival in comparison
to both the TF12 and untreated control groups (P<0.0059;
log-rank) (FIG. 18, FIG. 19). All the groups except the combination
of (E1)-3s plus PEGASYS.RTM. reached their respective MSTs. No
animals were euthanized for disease progression (TV>1.0
cm.sup.3) in this combination group. Importantly, the combination
of (E1)-3s plus PEGASYS.RTM. provided a significant survival
benefit when compared to all other treatments (P<0.0007;
log-rank) (FIG. 18, FIG. 19).
Example 5
Effect of Interferon-.alpha. Combination Therapy with
T-Cell-Redirecting Bispecific Antibodies in Human Gastric
Cancer
The methods and compositions disclosed in the preceding two
Examples were used to study the effects of leukocyte redirecting
bsAbs alone or in combination with interferon-.alpha.
(PEGASYS.RTM.) in the IFN-refractory NCI-N87 human gastric tumor
line. Groups of mice (N=8 each group) were injected s.c. with
5.times.10.sup.6 NCI-N87 cells+2.5.times.10.sup.6 T Cells (1:2 E:T
ratio) mixed with matrigel and therapy started 1 h later. The
treatment groups are shown in Table 10.
TABLE-US-00022 TABLE 10 Treatment Groups for (E1)-3s and TF12
Therapy (E1)-3s Therapy of a Human Gastric Carcinoma Xenograft
(NCI-N87) in NOD-SCID Mice Group Amount Injected Schedule 1
Untreated (NCI-N87 + T cells only) N.A. 2 (E1)-3s (47 .mu.g i.v.)
qdx5 3 TF12 (57 .mu.g i.v.) qdx5 4 PEGASYS .RTM. (0.6 .mu.g s.c.)
qwkx4 5 TF12 + PEGASYS .RTM. qdx5 + qwkx4 6 (E1)-3s + PEGASYS .RTM.
qdx5 + qwkx4
The effects of leukocyte redirecting bsAb (E1)-3s alone or in
combination with interferon are shown in FIG. 20 and FIG. 21. The
(E1)-3s bsAb was effective to reduce tumor growth and increase
survival in gastric cancer. Significantly, the combination with
interferon-.alpha. enhanced the effect of leukocyte redirecting
bsAb, even in an interferon resistant tumor. The combination
therapy was more effective than either agent added alone. Controls
with mice treated with TF12 bsAb alone or in combination with
interferon-.alpha. showed little effect on tumor growth or
mortality, compared to untreated animals.
Example 6
In Vivo Therapeutic Use of Antibody-Drug Conjugates (ADCs) in
Preclinical Models of Human Pancreatic or Colon Carcinoma
CL2A-SN-38-antibody conjugates were prepared as previously
described (see, e.g., U.S. Pat. Nos. 7,999,083 and 8,080,250).
Immune-compromised athymic nude mice (female), bearing subcutaneous
human pancreatic or colon tumor xenografts were treated with either
specific CL2A-SN-38 conjugate or control conjugate or were left
untreated. The therapeutic efficacies of the specific conjugates
were observed. In a Capan 1 pancreatic tumor model, specific
CL2A-SN-38 conjugates of hRS7 (anti-TROP2), hPAM4 (anti-MUC5ac),
and hMN-14 (anti-CEACAM5) antibodies showed better efficacies than
control hA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control
(not shown). Similarly in a BXPC3 model of human pancreatic cancer,
the specific hRS7-CL2A-SN-38 showed better therapeutic efficacy
than control treatments (not shown). Likewise, in an aggressive
LS174T model of human colon carcinoma, treatment with specific
hMN-14-CL2A-SN-38 was more efficacious than non-treatment (not
shown).
Example 7
In Vivo Therapy of Lung Metastases of GW-39 Human Colonic Tumors in
Nude Mice Using ADC hMN-14-[CL2-SN-38], IMMU-130
A lung metastatic model of colonic carcinoma was established in
nude mice by i.v. injection of GW-39 human colonic tumor
suspension, and therapy was initiated 14 days later. Specific
anti-CEACAM5 antibody conjugate, hMN14-CL2-SN-38, as well as
nontargeting anti-CD22 MAb control conjugate, hLL2-CL2-SN-38 and
equidose mixtures of hMN14 and SN-38 were injected at a dose
schedule of q4d.times.8, using different doses. Selective
therapeutic effects were observed with the hMN-14 ADC (not shown).
At a dosage of 250 .mu.g, the mice treated with hMN14-CL2-SN-38
showed a median survival of greater than 107 days. Mice treated
with the control conjugated antibody hLL2-CL2-SN-38, which does not
specifically target lung cancer cells, showed median survival of 77
days, while mice treated with unconjugated hMN14 IgG and free SN-38
showed a median survival of 45 days, comparable to the untreated
saline control of 43.5 days. A significant and surprising increase
in effectiveness of the conjugated, cancer cell targeted
antibody-SN-38 conjugate, which was substantially more effective
than unconjugated antibody and free chemotherapeutic agent alone,
was clearly seen (not shown). The dose-responsiveness of
therapeutic effect of conjugated antibody was also observed (not
shown). These results demonstrate the clear superiority of the
SN-38-antibody conjugate compared to the combined effect of both
unconjugated antibody and free SN-38 in the same in vivo human lung
cancer system.
Example 8
Use of ADC (IMMU-132 or hRS7-SN-38) to Treat Therapy-Refractive
Metastatic Colonic Cancer (mCRC)
The patient was a 62-year-old woman with mCRC who originally
presented with metastatic disease in January 2012. She had
laparoscopic ileal transverse colectomy as the first therapy a
couple of weeks after diagnosis, and then received 4 cycles of
FOLFOX (leucovorin, 5-fluorouracil, oxaliplatin) chemotherapy in a
neoadjuvant setting prior to right hepatectomy in March 2012 for
removal of metastatic lesions in the right lobe of the liver. This
was followed by an adjuvant FOLFOX regimen that resumed in June,
2012, for a total of 12 cycles of FOLFOX. In August, oxaliplatin
was dropped from the regimen due to worsening neurotoxicity. Her
last cycle of 5-FU was on 09/25/12.
CT done in January 2013 showed metastases to liver. She was then
assessed as a good candidate for enrollment to IMMU-132
(hRS7-SN-38) investigational study. Comorbidities in her medical
history include asthma, diabetes mellitus, hypertension,
hypercholesteremia, heart murmur, hiatal hernia, hypothyroidism,
carpel tunnel syndrome, glaucoma, depression, restless leg
syndrome, and neuropathy. Her surgical history includes
tubo-ligation (1975), thyroidectomy (1983), cholescystectomy
(2001), carpel tunnel release (2008), and glaucoma surgery.
At the time of entry into this therapy, her target lesion was a
3.1-cm tumor in the left lobe of the liver. Non-target lesions
included several hypo-attenuated masses in the liver. Her baseline
CEA was 781 ng/mL.
IMMU-132 was given on a once-weekly schedule by infusion for 2
consecutive weeks, then a rest of one week, this constituting a
treatment cycle. These cycles were repeated as tolerated. The first
infusion of IMMU-132 (8 mg/kg) was started on Feb. 15, 2013, and
completed without notable events. She experienced nausea (Grade 2)
and fatigue (Grade 2) during the course of the first cycle and has
been continuing the treatment since then without major adverse
events. She reported alopecia and constipation in March 2013. The
first response assessment done (after 6 doses) on Apr. 8, 2013
showed a shrinkage of target lesion by 29% by computed tomography
(CT). Her CEA level decreased to 230 ng/mL on Mar. 25, 2013. In the
second response assessment (after 10 doses) on May 23, 2013, the
target lesion shrank by 39%, thus constituting a partial response
by RECIST criteria. She has been continuing treatment, receiving 6
cycles constituting 12 doses of hRS7-SN-38 (IMMU-132) at 8 mg/kg.
Her overall health and clinical symptoms improved considerably
since starting this investigational treatment.
Example 9
ADC Therapy with IMMU-132 for Metastatic Solid Cancers
IMMU-132 is an ADC comprising the active metabolite of CPT-11,
SN-38, conjugated by a pH-sensitive linker (average drug-antibody
ratio=7.6) to the hRS7 anti-Trop-2 humanized monoclonal antibody,
which exhibits rapid internalization when bound to Trop-2. IMMU-132
targets Trop-2, a type I transmembrane protein expressed in high
prevalence and specificity by many carcinomas. This Example reports
a Phase I clinical trial of 25 patients with different metastatic
cancers (pancreatic, 7; triple-negative breast [TNBC], 4;
colorectal [CRC], 3; gastric, 3, esophageal, prostatic, ovarian,
non-small-cell lung, small-cell lung [SCLC], renal, tonsillar,
urinary bladder, 1 each) after failing a median of 3 prior
treatments (some including topoisomerase-I and -II inhibiting
drugs).
IMMU-132 was administered in repeated 21-day cycles, with each
treatment given on days 1 and 8. Dosing started at 8 mg/kg/dose
(i.e., 16 mg/kg/cycle), and escalated to 18 mg/kg before
encountering dose-limiting neutropenia, in a 3+3 trial design.
Fatigue, alopecia, and occasional mild to moderate diarrhea were
some of the more common non-hematological toxicities, with 2
patients also reporting a rash. Over 80% of 24 assessable patients
had stable disease or tumor shrinkage (SD and PR) among the various
metastatic cancers as best response by CT. Three patients (CRC,
TNBC, SCLC) have PRs by RECIST; median TTP for all patients,
excluding those with pancreatic cancer, is >18 weeks.
Neutropenia has been controlled by dose reduction to 8-10
mg/kg/dose (16-20 mg/kg/cycle).
Immunohistochemistry showed strong expression of Trop-2 in most
archived patient tumors, but it is not detected in serum.
Corresponding reductions in blood tumor marker titers (e.g., CEA,
CA19-9) reflected tumor responses. No anti-antibody or anti-SN-38
antibodies have been detected despite repeated dosing. Peak and
trough assessments of IMMU-132 concentrations in the serum show
that the conjugate clears completely within 7 days, an expected
finding based on in vitro studies showing 50% of the SN-38 is
released in the serum every day. These results indicate that this
novel ADC, given in doses ranging from 16-24 mg/kg per cycle, shows
a high therapeutic index in diverse metastatic solid cancers.
Example 10
IMMU-130, an SN-38 ADC that Targets CEACAM5, is Therapeutically
Active in Metastatic Colorectal Cancer (mCRC)
IMMU-130, an ADC of SN-38 conjugated by a pH-sensitive linker (7.6
average drug-antibody ratio) to the humanized anti-CEACAM5 antibody
(labetuzumab), is completing two Phase I trials. In both, eligible
patients with advanced mCRC were required to have failed/relapsed
standard treatments, one being the topoisomerase-I inhibiting drug,
CPT-11 (irinotecan), and an elevated plasma CEA (>5 ng/mL).
IMMU-130 was administered every 14 days (EOW) at doses starting
from 2.0 mg/kg in the first protocol (IMMU-130-01). Febrile
neutropenia occurred in 2 of 3 patients at 24 mg/kg; otherwise at
.ltoreq.16 mg/kg, neutropenia (.gtoreq.Grade 2) was observed in 7
patients, with one also experiencing thrombocytopenia. One patient
[of 8 who received .gtoreq.4 doses (2 cycles)] showed a 40.6%
decrease in liver (starting at 7 cm) and lung target lesions (PR by
RECIST) for 4.7 months, with no major toxicity, tolerating a total
of 18 doses at 16 mg/kg. The study is continuing at 12 mg/kg
EOW.
Since SN-38 is most effective in S-phase cells, a more protracted
exposure could improve efficacy. Thus, in a second Phase I trial
(IMMU-130-02), dosing was intensified to twice-weekly, starting at
6 mg/kg/dose for 2 weeks (4 doses) with 1 week off, as a treatment
cycle, in a 3+3 trial design. Neutropenia and manageable diarrhea
were the major side effects, until dose reduction to 4.0 mg/kg
twice-weekly, with early results indicating multiple cycles are
well-tolerated. Currently, tumor shrinkage occurred in 3 patients,
with 1 in continuing PR (-46%) by RECIST, among 6 patients who
completed .gtoreq.4 doses (1 cycle). In both trials, CEA blood
titers correlated with tumor response, and high levels did not
interfere with therapy. There have been no anti-antibody or
anti-SN-38 antibody reactions, based on ELISA tests. In each study,
the ADC was cleared by 50% within the first 24 h, which is much
longer exposure than with typical doses of the parental molecule,
CPT-11. These results indicate that this novel ADC, given in
different regimens averaging .about.16-24 mg/kg/cycle, shows a high
therapeutic index in advanced mCRC patients. Since CEACAM5 has
elevated expression in breast and lung cancers, as well as other
epithelial tumors, it may be a useful target in other cancers as
well.
Example 11
Antitumor Activity of Checkpoint Inhibitor Antibody Alone or
Combined with Anti-Trop-2.times.anti-CD3 bsAb, IFN-.alpha. or
Anti-Trop-2 ADC
To determine if the antitumor activity of the exemplary checkpoint
inhibitor antibody, ipilimumab (anti-CTLA4) is synergistic with or
inhibited by the addition of other therapeutic agents, CTLA4 mAb is
evaluated alone or in combination with the exemplary T-cell
redirecting bsAb (E1)-3s, with interferon-.alpha.
(PEGINTERFERON.RTM.), or with the exemplary ADC hRS7-SN-38
(IMMU-132) in murine tumor models. M109 lung carcinoma, SA1N
fibrosarcoma, and CT26 colon carcinoma models are chosen based on
different sensitivity to the various agents and CTLA4 blockade.
Human T cells are co-administered with the antibodies.
All compounds are tested at their optimal dose and schedule. When
used in combination, CTLA4 mAb is initiated one day after the first
dose of IMMU-132, (E1)-3s or interferon-.alpha.. Percent tumor
growth inhibition and number of days to reach target tumor size are
used to evaluate efficacy. Antitumor activity is scored as:
complete regression (CR; non-palpable tumor) or partial regression
(PR; 50% reduction in tumor volume). Synergy is defined as
antitumor activity significantly superior (p<0.05) to the
activity of monotherapy with each agent.
In the SA1N fibrosarcoma tumor model, which is sensitive to CTLA4
blockade and modestly sensitive to (E1)-3s, interferon-.alpha., and
IMMU-132, borderline synergy is evident with the combination of
CTLA4 mAb and (E1)-3s, whereas no effect is observed with
interferon-.alpha.. IMMU-132 monotherapy does not produce
significant SA1N antitumor activity. However, combining IMMU-132
with CTLA4 mAb results in synergy. In the M109 lung metastasis
model and CT26 colon carcinoma model, synergy is detected for CTLA4
mAb combined with each of IMMU-132, (E1)-3s and
interferon-.alpha..
In summary, addition of CTLA4 mAb to interferon-.alpha., IMMU-132,
or (E1)-3s results in model-dependent synergistic activities.
Synergy is observed regardless of the immunogenicity of the tumor
and only when at least one of the therapies is active. All
combination regimens are well-tolerated and the combination
therapies do not appear to inhibit CTLA4 mAb activity. Synergy is
observed in tumors unresponsive to CTLA4 mAb alone, suggesting that
the other therapeutic agents might induce immunogenic cell
death.
Example 12
Combination Therapy with Anti-Trop-2 ADC (IMMU-132) and
Interferon-.alpha. (PEGINTERFERON.RTM.) to Treat Refractory,
Metastatic, Non-Small Cell Lung Cancer
The patient is a 60-year-old man diagnosed with non-small cell lung
cancer. The patient is given chemotherapy regimens of carboplatin,
bevacizumab for 6 months and shows a response, and then after
progressing, receives further courses of chemotherapy with
carboplatin, etoposide, TAXOTERE.RTM., gemcitabine over the next 2
years, with occasional responses lasting no more than 2 months. The
patient then presents with a left mediastinal mass measuring
6.5.times.4 cm and pleural effusion.
After signing informed consent, the patient is given IMMU-132 at a
dose of 18 mg/kg every other week. After the first week of
treatment, the patient is given combination therapy with IMMU-132
and PEGINTERFERON.RTM.. During the first two injections, brief
periods of neutropenia and diarrhea are experienced, with 4 bowel
movements within 4 hours, but these resolve or respond to
symptomatic medications within 2 days. After a total of 6 infusions
of IMMU-132 and 5 infusions of PEGINTERFERON.RTM., CT evaluation of
the index lesion shows a 22% reduction, just below a partial
response but definite tumor shrinkage. The patient continues with
this therapy for another two months, when a partial response of 45%
tumor shrinkage of the sum of the diameters of the index lesion is
noted by CT, thus constituting a partial response by RECIST
criteria. The combination therapy appears to provide a synergistic
response, compared to the two agents administered separately.
Example 13
Combination Therapy with ADC (IMMU-130) and T-Cell Redirecting bsAb
(MT100) to Treat Advanced Colonic Cancer
The patient is a 75-year-old woman initially diagnosed with
metastatic colonic cancer (Stage IV). She has a right partial
hemicolectomy and resection of her small intestine and then
receives FOLFOX, FOLFOX+bevacizumab, FOLFIRI+ramucirumab, and
FOLFIRI+cetuximab therapies for a year and a half, when she shows
progression of disease, with spread of disease to the posterior
cul-de-sac, omentum, with ascites in her pelvis and a pleural
effusion on the right side of her chest cavity. Her baseline CEA
titer just before this therapy is 15 ng/mL. She is given 6 mg/kg
IMMU-130 (anti-CEACAM5-SN-38) twice weekly for 2 consecutive weeks,
and then one week rest (3-week cycle). After the first cycle, the
patient is given combination therapy with IMMU-132 and the
leukocyte redirecting bsAb MT110, which is administered by
continuous infusion on the same 3-week cycle. After 5 cycles, which
are tolerated very well, without any major hematological or
non-hematological toxicities, her plasma CEA titer shrinks modestly
to 1.3 ng/mL, but at the 8-week evaluation she shows a 21%
shrinkage of the index tumor lesions, which increases to a 27%
shrinkage at 13 weeks. Surprisingly, the patient's ascites and
pleural effusion both decrease (with the latter disappearing) at
this time, thus improving the patient's overall status remarkably.
The combination therapy appears to provide a synergistic response,
compared to the two agents administered separately.
Example 14
Combination Therapy with ADC (IMMU-130), Anti-Trop-2.times.Anti-CD3
bsAb ((E1)-3s) and Interferon-.alpha. to Treat Gastric Cancer
Patient with Stage IV Metastatic Disease
The patient is a 52-year-old male who sought medical attention
because of gastric discomfort and pain related to eating for about
6 years, and with weight loss during the past 12 months. Palpation
of the stomach area reveals a firm lump which is then gastroscoped,
revealing an ulcerous mass at the lower part of his stomach. This
is biopsied and diagnosed as a gastric adenocarcinoma. Laboratory
testing reveals no specific abnormal changes, except that liver
function tests, LDH, and CEA are elevated, the latter being 10.2
ng/mL. The patent then undergoes a total-body PET scan, which
discloses, in addition to the gastric tumor, metastatic disease in
the left axilla and in the right lobe of the liver (2 small
metastases). The patient has his gastric tumor resected, and then
has baseline CT measurements of his metastatic tumors. Four weeks
after surgery, he receives 3 courses of combination chemotherapy
consisting of a regimen of cisplatin and 5-fluorouracil (CF), but
does not tolerate this well, so is switched to treatment with
docetaxel. It appears that the disease is stabilized for about 4
months, based on CT scans, but then the patient's complaints of
further weight loss, abdominal pain, loss of appetite, and extreme
fatigue cause repeated CT studies, which show increase in size of
the metastases by a sum of 20% and a suspicious lesion at the site
of the original gastric resection.
The patient is then given experimental therapy with IMMU-130
(anti-CEACAM5-SN-38) on a weekly schedule of 8 mg/kg. After the
first week, combination therapy with IMMU-130, (E1)-3s and
interferon-.alpha. is initiated. The patient exhibits no evidence
of diarrhea or neutropenia over the following 4 weeks. The patient
then undergoes a CT study to measure his metastatic tumor sizes and
to view the original area of gastric resection. The radiologist
measures, according to RECIST criteria, a decrease of the sum of
the metastatic lesions, compared to baseline prior to therapy, of
23%. There does not seem to be any clear lesion in the area of the
original gastric resection. The patient's CEA titer at this time is
7.2 ng/mL, which is much reduced from the baseline value of 14.5
ng/mL. The patient continues on weekly combination therapy, and
after a total of 13 infusions, his CT studies show that one liver
metastasis has disappeared and the sum of all metastatic lesions is
decreased by 41%, constituting a partial response by RECIST. The
patient's general condition improves and he resumes his usual
activities while continuing to receive maintenance therapy every
third week. At the last measurement of blood CEA, the value is 4.8
ng/mL, which is within the normal range for a smoker, which is the
case for this patient.
Example 15
General Techniques for Dock-And-Lock.TM.
The general techniques discussed below may be used to generate
DNL.TM. complexes with AD or DDD moieties attached to any
antibodies or antigen-binding antibody fragments, using the
disclosed methods and compositions.
Expression Vectors
The plasmid vector pdHL2 has been used to produce a number of
antibodies and antibody-based constructs. See Gillies et al., J
Immunol Methods (1989), 125:191-202; Losman et al., Cancer (Phila)
(1997), 80:2660-6. The di-cistronic mammalian expression vector
directs the synthesis of the heavy and light chains of IgG. The
vector sequences are mostly identical for many different IgG-pdHL2
constructs, with the only differences existing in the variable
domain (V.sub.H and V.sub.L) sequences. Using molecular biology
tools known to those skilled in the art, these IgG expression
vectors can be converted into Fab-DDD or Fab-AD expression
vectors.
To generate Fab-DDD expression vectors, the coding sequences for
the hinge, CH2 and CH3 domains of the heavy chain were replaced
with a sequence encoding the first 4 residues of the hinge, a 14
residue linker and a DDD moiety, such as the first 44 residues of
human RII.alpha. (referred to as DDD1, SEQ ID NO:1). To generate
Fab-AD expression vectors, the sequences for the hinge, CH2 and CH3
domains of IgG were replaced with a sequence encoding the first 4
residues of the hinge, a 15 residue linker and an AD moiety, such
as a 17 residue synthetic AD called AKAP-IS (referred to as AD1,
SEQ ID NO:3), which was generated using bioinformatics and peptide
array technology and shown to bind RII.alpha. dimers with a very
high affinity (0.4 nM). See Alto, et al. Proc. Natl. Acad. Sci.,
U.S.A (2003), 100:4445-50. Two shuttle vectors were designed to
facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD1
or Fab-AD1 expression vectors, as described below.
Preparation of CH1
The CH1 domain was amplified by PCR using the pdHL2 plasmid vector
as a template. The left PCR primer consisted of the upstream (5')
end of the CH1 domain and a SacII restriction endonuclease site,
which is 5' of the CH1 coding sequence. The right primer consisted
of the sequence coding for the first 4 residues of the hinge (PKSC,
SEQ ID NO:102) followed by four glycines and a serine, with the
final two codons (GS) comprising a Bam HI restriction site. The 410
bp PCR amplimer was cloned into the PGEMT.RTM. PCR cloning vector
(PROMEGA.RTM., Inc.) and clones were screened for inserts in the T7
(5') orientation.
A duplex oligonucleotide was synthesized to code for the amino acid
sequence of DDD1 preceded by 11 residues of the linker peptide,
with the first two codons comprising a BamHI restriction site. A
stop codon and an EagI restriction site are appended to the 3'end.
The encoded polypeptide sequence is shown below.
TABLE-US-00023 (SEQ ID NO: 103)
GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA
Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom,
which overlap by 30 base pairs on their 3' ends, were synthesized
and combined to comprise the central 154 base pairs of the 174 bp
DDD1 sequence. The oligonucleotides were annealed and subjected to
a primer extension reaction with Taq polymerase. Following primer
extension, the duplex was amplified by PCR. The amplimer was cloned
into PGEMT.RTM. and screened for inserts in the T7 (5')
orientation.
A duplex oligonucleotide was synthesized to code for the amino acid
sequence of AD1 preceded by 11 residues of the linker peptide with
the first two codons comprising a BamHI restriction site. A stop
codon and an EagI restriction site are appended to the 3'end. The
encoded polypeptide sequence is shown below.
TABLE-US-00024 (SEQ ID NO: 104) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA
Two complimentary overlapping oligonucleotides encoding the above
peptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, were
synthesized and annealed. The duplex was amplified by PCR. The
amplimer was cloned into the PGEMT.RTM. vector and screened for
inserts in the T7 (5') orientation.
Ligating DDD1 with CH1
A 190 bp fragment encoding the DDD1 sequence was excised from
PGEMT.RTM. with BamHI and NotI restriction enzymes and then ligated
into the same sites in CH1-PGEMT.RTM. to generate the shuttle
vector CH1-DDD1-PGEMT.RTM..
Ligating AD1 with CH1
A 110 bp fragment containing the AD1 sequence was excised from
PGEMT.RTM. with BamHI and NotI and then ligated into the same sites
in CH1-PGEMT.RTM. to generate the shuttle vector
CH1-AD1-PGEMT.RTM..
With this modular design either CH1-DDD1 or CH1-AD1 can be
incorporated into any IgG construct in the pdHL2 vector. The entire
heavy chain constant domain is replaced with one of the above
constructs by removing the SacII/EagI restriction fragment
(CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment
of CH1-DDD1 or CH1-AD1, which is excised from the respective
PGEMT.RTM. shuttle vector.
C-DDD2-Fd-hMN-14-pdHL2
C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of
C-DDD2-Fab-hMN-14, which possesses a dimerization and docking
domain sequence of DDD2 (SEQ ID NO:2) appended to the carboxyl
terminus of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser
peptide linker. The fusion protein secreted is composed of two
identical copies of hMN-14 Fab held together by non-covalent
interaction of the DDD2 domains.
The expression vector was engineered as follows. Two overlapping,
complimentary oligonucleotides, which comprise the coding sequence
for part of the linker peptide and residues 1-13 of DDD2, were made
synthetically. The oligonucleotides were annealed and
phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3'
ends that are compatible for ligation with DNA digested with the
restriction endonucleases BamHI and PstI, respectively.
The duplex DNA was ligated with the shuttle vector
CH1-DDD1-PGEMT.RTM., which was prepared by digestion with BamHI and
PstI, to generate the shuttle vector CH1-DDD2-PGEMT.RTM.. A 507 bp
fragment was excised from CH1-DDD2-PGEMT.RTM. with SacII and EagI
and ligated with the IgG expression vector hMN-14(I)-pdHL2, which
was prepared by digestion with SacII and EagI. The final expression
construct was designated C-DDD2-Fd-hMN-14-pdHL2. Similar techniques
have been utilized to generated DDD2-fusion proteins of the Fab
fragments of a number of different humanized antibodies.
h679-Fd-AD2-pdHL2
h679-Fab-AD2, was designed to pair to C-DDD2-Fab-hMN-14.
h679-Fd-AD2-pdHL2 is an expression vector for the production of
h679-Fab-AD2, which possesses an anchoring domain sequence of AD2
(SEQ ID NO:4) appended to the carboxyl terminal end of the CH1
domain via a 14 amino acid residue Gly/Ser peptide linker. AD2 has
one cysteine residue preceding and another one following the anchor
domain sequence of AD1.
The expression vector was engineered as follows. Two overlapping,
complimentary oligonucleotides (AD2 Top and AD2 Bottom), which
comprise the coding sequence for AD2 and part of the linker
sequence, were made synthetically. The oligonucleotides were
annealed and phosphorylated with T4 PNK, resulting in overhangs on
the 5' and 3' ends that are compatible for ligation with DNA
digested with the restriction endonucleases BamHI and SpeI,
respectively.
The duplex DNA was ligated into the shuttle vector
CH1-AD1-PGEMT.RTM., which was prepared by digestion with BamHI and
SpeI, to generate the shuttle vector CH1-AD2-PGEMT.RTM.. A 429 base
pair fragment containing CH1 and AD2 coding sequences was excised
from the shuttle vector with SacII and EagI restriction enzymes and
ligated into h679-pdHL2 vector that prepared by digestion with
those same enzymes. The final expression vector is
h679-Fd-AD2-pdHL2.
Generation of TF2 DNL.TM. Construct
A trimeric DNL.TM. construct designated TF2 was obtained by
reacting C-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2
was generated with >90% yield as follows. Protein L-purified
C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-AD2 (60 mg) at a
1.4:1 molar ratio. The total protein concentration was 1.5 mg/ml in
PBS containing 1 mM EDTA. Subsequent steps involved TCEP reduction,
HIC chromatography, DMSO oxidation, and IMP 291 affinity
chromatography. Before the addition of TCEP, SE-HPLC did not show
any evidence of a.sub.2b formation. Addition of 5 mM TCEP rapidly
resulted in the formation of a.sub.2b complex consistent with a 157
kDa protein expected for the binary structure. TF2 was purified to
near homogeneity by IMP 291 affinity chromatography (not shown).
IMP 291 is a synthetic peptide containing the HSG hapten to which
the 679 Fab binds (Rossi et al., 2005, Clin Cancer Res
11:7122s-29s). SE-HPLC analysis of the IMP 291 unbound fraction
demonstrated the removal of a.sub.4, a.sub.2 and free kappa chains
from the product (not shown).
The functionality of TF2 was determined by BIACORE.RTM. assay. TF2,
C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent
a.sub.2b complex), or C-DDD2-hMN-14+h679-AD2 (used as a control
sample of unreduced a.sub.2 and b components) were diluted to 1
.mu.g/ml (total protein) and passed over a sensorchip immobilized
with HSG. The response for TF2 was approximately two-fold that of
the two control samples, indicating that only the h679-Fab-AD
component in the control samples would bind to and remain on the
sensorchip. Subsequent injections of WI2 IgG, an anti-idiotype
antibody for hMN-14, demonstrated that only TF2 had a
DDD-Fab-hMN-14 component that was tightly associated with
h679-Fab-AD as indicated by an additional signal response. The
additional increase of response units resulting from the binding of
WI2 to TF2 immobilized on the sensorchip corresponded to two fully
functional binding sites, each contributed by one subunit of
C-DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind
two Fab fragments of WI2 (not shown).
Production of TF10 DNL.TM. Construct
A similar protocol was used to generate a trimeric TF10 DNL.TM.
construct, comprising two copies of a C-DDD2-Fab-hPAM4 and one copy
of C-AD2-Fab-679. The TF10 bispecific ([hPAM4].sub.2.times.h679)
antibody was produced using the method disclosed for production of
the (anti CEA).sub.2.times.anti HSG bsAb TF2, as described above.
The TF10 construct bears two humanized PAM4 Fabs and one humanized
679 Fab.
The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressed
independently in stably transfected myeloma cells. The tissue
culture supernatant fluids were combined, resulting in a two-fold
molar excess of hPAM4-DDD2. The reaction mixture was incubated at
room temperature for 24 hours under mild reducing conditions using
1 mM reduced glutathione. Following reduction, the reaction was
completed by mild oxidation using 2 mM oxidized glutathione. TF10
was isolated by affinity chromatography using IMP291-affigel resin,
which binds with high specificity to the h679 Fab.
Example 16
Production of AD- and DDD-Linked Fab and IgG Fusion Proteins from
Multiple Antibodies
Using the techniques described in the preceding Example, the IgG
and Fab fusion proteins shown in Table 11 were constructed and
incorporated into DNL.TM. constructs. The fusion proteins retained
the antigen-binding characteristics of the parent antibodies and
the DNL.TM. constructs exhibited the antigen-binding activities of
the incorporated antibodies or antibody fragments.
TABLE-US-00025 TABLE 11 Fusion proteins comprising IgG or Fab
Binding Fusion Protein Specificity C-AD1-Fab-h679 HSG
C-AD2-Fab-h679 HSG C-(AD).sub.2-Fab-h679 HSG C-AD2-Fab-h734 Indium-
DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20 C-AD2-Fab-hL243
HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22 C-AD2-IgG-hMN-14
CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1 C-AD2-IgG-hPAM4
MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5 C-DDD2-Fab-hMN-14
CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19 C-DDD2-Fab-hA20
CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DR C-DDD2-Fab-hLL1
CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6
C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1
IGF-1R C-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5
Example 17
Use of NK-Targeted Leukocyte-Redirecting bsAbs
The use of bsAbs to retarget leukocytes is not limited to
antibodies against T cells. In alternative embodiments, bsAbs that
bind to monocytes, NK cells or neutrophils may also be used for
retargeting purposes.
CD16 is an activating low-affinity Fc-.gamma. receptor for IgG,
which is highly expressed by the CD56.sup.dim subset of NK cells
(Gleason et al., 2012, Mol Cancer Ther 11:2674-84). In addition to
their use in NK cell retargeting, bsAbs comprising an anti-CD16
antibody component have the ability to activate NK-mediated
cytotoxicity through direct signaling of CD16, inducing directed
secretion of lytic granules and target cell death (Gleason et al.,
2012).
A CD16/CD19 bispecific killer cell engager (BiKE) and a
CD16/CD19/CD22 trispecific killer cell engager (TriKe) are prepared
according to (Gleason et al., 2012, Mol Cancer Ther 11:2674-84),
using DNA shuffling and ligation techniques as previously reported
(Vallera et al., 2005, Clin Cancer Res 11:3879-88). The expressed
BiKE and TriKE are purified by sequential ion exchange and
size-exclusion column chromatography. Resting PBMCs are exposed to
primary ALL and CLL tumor cells in the presence of CD16/CD19 BiKE
or CD16/CD19/CD22 TriKE (10 .mu.g/mL). A significant increase in
cytotoxicity to tumor cells is observed in the presence of the BiKE
or TriKE, compared to cells without retargeting antibody. No effect
is observed on tumor cells exposed to BiKE or TriKE in the absence
of PBMCs. The TriKE has a greater effect on tumor cell toxicity
relative to the BiKE, indicating that binding to an additional
tumor cell antigen may enhance the retargeting effect. Similar
results are obtained using purified NK cells instead of PBMCs.
A CD16/CD33 BiKE is prepared as disclosed in Wiernik et al. (2013,
Clin Cancer Res 19:3844-55. The BiKE is administered to nude mice
injected with human HL60 promyelocytic leukemia xenograft cells,
co-administered with human PBMCs. The BiKE treated mice show a
decreased mortality and tumor growth rate compared to mice treated
with control bsAbs. Addition of an anti-CD33-SN-38 ADC further
enhances the cytotoxic effect of the BiKE.
Example 18
Trivalent Antibodies for Therapeutic Use
A trivalent, trispecific cell targeting construct is made as
described in patent EP1309795B1 comprising: (i) chimerizing or
humanizing a mouse anti-CD16 mab as described in U.S. Pat. No.
618,728 from which the Fab of Claim 1 of EP1309795 is derived; (ii)
constructing a single chain antibody comprised of the Fv of the
humanized anti-Trop-2 antibody described in U.S. Pat. No.
7,238,785, and joining the scFv by a linker to the carboxyl
terminal of the light chain of the anti-CD16 Fab of (i); and (iii)
constructing a single chain of the Fv of the humanized anti-CD19
described in U.S. Pat. No. 8,486,395 and joining the scFv by a
linker to the carboxyl terminal of the CH1 of the anti-CD16 Fab of
(ii).
The trivalent construct is administered to a subject with
metastatic pancreatic cancer, in combination with IMMU-132. A
partial response is observed and the tumor shows a regression in
size that lasts for 12 months.
Example 19
Anti-Trop-2.times.Anti-CD3 Bispecific Antibody
A bispecific antibody (bsAb) was produced as a tandem single-chain
variable fragment (scFv) for redirecting T cells via CD3 binding to
tumor cells, particularly carcinomas, via Trop-2 targeting. Trop-2
is a tumor-associated antigen (TAA) that could be highly effective
for targeting various epithelial cancers. However, it has yet to be
investigated in any bsAb format for T-cell-redirected therapy.
Trop-2 is a 35 kDa transmembrane glycoprotein that is overexpressed
relative to normal tissues in a variety of human cancers, including
pancreatic and gastric carcinomas, where increased expression
correlates with poor prognosis (Fong et al., 2008, Br J Cancer
99:1290-5; Iacobuzio-Donahue et al., 2002, Am J Pathol 160:1239-49;
Kapoor, 2013, Tumour Biol 34:1967-8; Muhlmann et al., 2009, J Clin
Pathol 62:152-8; Stein et al., 1993, Int J Cancer 55:938-46; Stein
et al., 1993, Int J Cancer 55:938-46). Variable domains (VH and VK)
derived from hRS7, the humanized version of the original murine
anti-Trop-2 mAb, RS7, were combined with the variable domains of
the murine anti-CD3 mAb, Okt3, to generate the E1-3 bsAb.
Construction of a Plasmid Vector for Expression of E1-3 in
Mammalian Cells
A double stranded DNA sequence (SEQ ID NO:106) was synthesized and
assembled into the pUC57 plasmid vector. SEQ ID NO:106 was excised
from pUC57 by digestion with Xba I and Eag I restriction
endonucleases, and ligated into the pdHL2 mammalian expression
vector, which was prepared by digestion with the same enzymes. The
coding sequence directs the synthesis of a single polypeptide (SEQ
ID NO:107) comprising a leader peptide, hRS7VK (SEQ ID NO:108), L1
(SEQ ID NO:109), hRS7VH (SEQ ID NO:110), L2 (SEQ ID NO:111), Okt3VH
(SEQ ID NO:112), L3 (SEQ ID NO:113), Okt3VK (SEQ ID NO:114), and
6-His (SEQ ID NO:105). A schematic representation of the tandem
scFv E1-3 is shown in FIG. 22.
TABLE-US-00026 Synthetic DNA sequence comprising E1-3 insert (SEQ
ID NO: 106) tctagacacaggccgccatcatgggatggagctgtatcatcctcttctt
ggtagcaacagctacaggtgtccactccgacattcagctgacccagtct
ccatcctccctgtctgcatctgtaggagacagagtcagcatcacctgca
aggccagtcaggatgtgagtattgctgtagcctggtatcagcagaaacc
agggaaagcccctaagctcctgatctactcggcatcctaccggtacact
ggagtccctgataggttcagtggcagtggatctgggacagatttcactc
tcaccatcagcagtctgcaacctgaagattttgcagrnattactgtcag
caacattatattactccgctcacgttcggtgctgggaccaaggtggaga
tcaaaggtggaggagggtccggtggaggagggtctggtggaggagggag
ccaggtccagctgcagcaatctgggtctgagttgaagaagcctggggcc
tcagtgaaggrncctgcaaggcttctggatacaccttcacaaactatgg
aatgaactgggtgaagcaggcccctggacaagggcttaaatggatgggc
tggataaacacctacactggagagccaacatatactgatgacttcaagg
gacggtttgccttctccttggacacctctgtcagcacggcatatctcca
gatcagcagcctaaaggctgacgacactgccgtgtatttctgtgcaaga
ggggggttcggtagtagctactggtacttcgatgtctggggccaagggt
ccctggtcaccgtctcctcaggtggcggagggtccgatatcaagctgca
gcagtctggagcagagctcgctcgaccaggagctagtgtgaagatgtca
tgtaaaacaagtggctatactttcacccggtacactatgcactgggtca
agcagcgcccaggacagggtctggaatggatcggctacattaaccccag
caggggatataccaactacaatcagaagttcaaggataaagccaccctg
actaccgacaagtcctctagtacagcttatatgcagctgtcaagcctca
cttccgaggactctgcagtgtattactgcgccagatattacgacgatca
ttattgtctggattactggggccagggaacaactctcacagtgtcctct
gtcgaaggtggcagtggagggtcaggtggcagcggagggtccggtggag
tggacgatatccagctgacccagtctcctgccattatgagcgcttcccc
aggcgagaaggtgacaatgacttgccgggccagttcaagcgtcagctat
atgaattggtatcagcagaagtctggaaccagtcctaaacgatggatct
atgacacatctaaagtggcaagcggggtcccatacaggttctctgggag
tggttcaggcactagctattccctgaccatttcctctatggaggccgaa
gatgcagccacctattactgtcagcagtggagttcaaatccactcacct
tcggagcaggcactaaactggaactcaagcaccaccaccaccaccacta aggcggccg Deduced
amino acid sequence of E1-3 (SEQ ID NO: 107)
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIY
SASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF
GAGTKVEIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKAS
GYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDT
SVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSGG
GGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLE
WIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYY
CARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQS
PAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASG
VPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLEL KHHHHHH Amino
acid sequence of hRS7 VK (SEQ ID NO: 108)
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIY
SASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF GAGTKVEIK Amino
acid sequence of linker L1 (SEQ ID NO: 109) GGGGSGGGGSGGGGS Amino
acid sequence hRS7 VH (SEQ ID NO: 110)
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG
WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR
GGFGSSYWYFDVWGQGSLVTVSS Amino acid sequence of linker L2 (SEQ ID
NO: 111) GGGGS Amino acid sequence of Okt3 VH (SEQ ID NO: 112)
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGY
INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY
DDHYCLDYWGQGTTLTVSS Amino acid sequence of linker L3 (SEQ ID NO:
113) VEGGSGGSGGSGGSGGVD Amino acid sequence of Okt3 VK (SEQ ID NO:
114) DIQLTQSPAIMSASPGEKVTMTCRASS SVSYMNWYQQKSGTSPKRWIYD
TSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGA GTKLE
Development of a Stable Production Clone in SpESF Myeloma Cells
The E1-3-pdHL2 vector was linearized by digestion with Sal I
restriction endonuclease and 30 .mu.g was used to stably transfect
1.times.10.sup.7 SpESFX myeloma cells (Rossi et al., 2011,
Biotechnol Prog 27:766-75) by electroporation using two pulses at
850 V and 10 .mu.F. Selection and production media was supplemented
with 0.2 .mu.M methotrexate (MTX). Transfectant clones were
selected in 96-well tissue culture plates and screened for E1-3
expression by ELISA using Ni-NTA 96-well plates. The E1-3 protein
was purified from the culture broth of roller bottle cultures by
immobilized metal affinity chromatography (IMAC) using
Nickel-SEPHAROSE.RTM. resin, followed by size exclusion high
performance liquid chromatography (SE-HPLC). The purified product
resolved as a single SE-HPLC peak (not shown) and a single
polypeptide band by SDS-PAGE (not shown), with relative mobilities
consistent with its calculated molecular size of 53,423 Da.
Example 20
Redirected T Cell Killing of Trop-2-Expressing Solid Tumor Cells Ex
Vivo
Peripheral blood mononuclear cells (PBMCs) were prepared from the
buffy coat of blood specimens of two healthy donors (Blood Center
of NJ), and used for the isolation of CD8.sup.+ T cells (Miltenyi).
Capan-1 (pancreatic cancer, 157,000 Trop-2/cell), BxPC3 (pancreatic
cancer, 500,000 Trop-2/cell) and NCI-N87 (gastric cancer, 247,000
Trop-2/cell) cell lines (ATCC) were used as target cells expressing
low-, high- and mid-levels of Trop-2. BxPC3 and NCI-N87 were
maintained in RPMI1640 media supplemented with 10% FBS, while
Capan-1 cells were maintained in 20% FBS/RPMI1640. CD8.sup.+ T
cells (1.2.times.10.sup.5 cells/well) were combined with target
cells (2.times.10.sup.4 cells/well) at a 6:1 ratio in 96-well
tissue culture plates. Titrations of E1-3 and (E1)-3s were added to
the assay plates. Following a 48-hour incubation at 37.degree. C.,
plates were washed twice with PBS to remove the T cells, and then
150 .mu.L of fresh media supplemented with 30% MTS reagent
(CELLTITER 96.RTM. Aqueous One Solution, Promega) was added to each
well. Absorbance at 490 nm was measured after 1-2 h at 37.degree.
C. with an ENVISION plate Reader.RTM..
The in vitro potency of the E1-3 bispecific antibody was compared
with that of the equivalent DNL construct, (E1)-3s, in three
Trop-2-expressing cell lines (BxPC3, Capan-1 and NCI-N87) using T
cells from three donors for each cell line (FIG. 23). Based on the
IC.sub.50 values (Table 12), E1-3 is at least 5-fold more potent
than (E1)-3s in all three cell lines, whose relative sensitivity to
E1-3 appears to correlate with the Trop-2-antigen density, when
compared with T cells from the same donor. However, potency was
varied among the donor T cells used. In vitro, E1-3 mediated a
highly potent T-cell lysis of BxPC3 [IC.sub.50=0.09(.+-.0.04) pM],
Capan-1 [IC.sub.50=1.2(.+-.1.1) pM] and NCI-N87
[IC.sub.50=1.2(.+-.1.2) pM] target cells.
TABLE-US-00027 TABLE 12 IC.sub.50 values for ex vivo T cell
redirected killing of Trop-2.sup.+ cancer cell lines with E1-3 and
(E1)-3s. BxPC3 Capan-1 NCI-N87 500,000/cell 157,000/cell
247,000/cell Trop-2 Donor 1 Donor 2 Donor 3 Donor 1 Donor 2 Donor 4
Donor 1 Donor 2 Donor 5 E1-3 0.12 0.10 0.05 0.58 2.7 0.47 0.29 0.76
2.50 (E1)-3s 1.06 0.56 0.32 35.6 248 8.51 6.76 34 NA* IC.sub.50
values = pM concentration resulting in 50% killing. *Did not
achieve 50% killing. Donors 1 and 2 were the same for each donor.
Donors 3, 4 and 5 were independent donors.
Example 21
In Vivo Therapy of Solid Tumors with E1-3 vs. (E1)-3s
Female 4-8-week old NOD/SCID mice were administered subcutaneous
injections of a mixture of PBMCs and NCI-N87 (2:1) mixed with an
equal volume of MATRIGEL.RTM.. Therapy consisted of i.v. injections
of 50 .mu.g of E1-3 on days 1 and 4, or daily injections with 47
.mu.g of (E1)-3s on days 1 through 5. The untreated group received
the mixture of NCI-N87 and PBMCs without bsAb. Tumor volume (TV)
was determined twice weekly by measurements in two dimensions using
calipers, with volumes defined as: L.times.W.sup.2/2, where L is
the longest dimension of the tumor and W the shortest (FIG. 24).
Statistical analysis of tumor growth was based on area under the
curve (AUC). Profiles of individual tumor growth were obtained
through linear-curve modeling. An F-test was employed to determine
equality of variance between groups prior to statistical analysis
of growth curves. A Critical Z test on the survival data identified
any outliers within a given treatment group with P<0.05 censored
from the final data analysis. A two-tailed t-test was used to
assess statistical significance between the various treatment
groups and controls, except for the untreated control, where a
one-tailed t-test was used. Additionally, efficacy was determined
by log-rank using Prism software on Kaplan-Meier curves using
survival surrogate endpoints as time for tumor progression (TTP) to
1.0 cm3. Significance was considered at P.ltoreq.0.05 for all
comparisons.
Both E1-3 (P) and (E1)-3s delayed growth of NCI-N87 tumors
significantly (P.ltoreq.0.001; AUC.sub.day25) (FIG. 24). The E1-3
was superior to (E1)-3s (P=0.0324, AUC.sub.day36) (FIG. 24). In
vivo, two 50-.mu.g doses of E1-3 given three days apart cured all
of the mice (N=8) bearing NCI-N87 xenografts (P=0.0005; Log-Rank).
Tumors in the control group (PBMCs only) reached the endpoint
(TV>1 cm.sup.3) at 39.5 days. All mice were tumor-free in the
E1-3 group after 78 days.
Example 22
Trogocytosis Induced by Anti-CD3.times.Anti-Trop-2 Bispecific
Antibodies
Trop-2 has limited presence on normal tissues but is highly
expressed in diverse epithelial cancers. As discussed in the
Examples above, (E1)-3s is a T-cell-redirecting trivalent
bispecific antibody (bsAb) DNL.RTM. complex, comprising an anti-CD3
scFv covalently linked to a stabilized dimer of a Trop-2-targeting
Fab. We show herein for the first time that bsAb-mediated
bidirectional trogocytosis occurs between target cells and T cells
and involves formation of immunologic synapses.
Methods
BxPC3 cells were detached with trypsin (which does not affect
Trop-2) and mixed with purified T cells. Cell mixtures were treated
with 0.1 nmol/L bsAbs at 37.degree. C. for 1 hour. Cells were
stained with either: (i) anti-Trop-2 MABC518 followed by GAM-FITC,
or (ii) anti-Trop-2-PE clone MR54 and anti-CD4-APC. Single BxPC3
and T cells were gated from cell conjugates by forward and side
scattering, as well as Trop-2 and CD4 fluorescence.
Results
(E1)-3s induces the formation of immunologic synapses between T
cells and target cells. This was shown using Capan-1 pancreatic
carcinoma cells (Rossi et al., 2013, MAbs 6:381-91). Here, addition
of 0.1 .mu.g/mL (E1)-3s to a mixture of purified CD8.sup.+ T cells
and NCI-N87 gastric carcinoma cells, which were membrane-labeled
with red and green fluorescence, respectively, resulted in the
formation of conjugates evident by fluorescence microscopy (not
shown). No conjugates were observed in the presence of (19)-3s (not
shown) or TF12 (not shown), which bind only T cells or NCI-N87,
respectively. Dunking the slides in saline washed off the vast
majority of T cells in wells containing (19)-3s or TF12, whereas
many T cells remained bound to the adherent NCI-N87 cells in the
wells treated with (E1)-3s.
Treatment of BxPC3 (500,000 Trop-2/cell) and purified T-cell
mixtures with (E1)-3s specifically induced trogocytosis, whereby
Trop-2 was transferred from BxPC3 to T cells (FIG. 25). Whereas
(E1)-3s treatment resulted in 40% Trop-2.sup.+ T cells, <5% of
the T cells were counted in the Trop-2.sup.+ gate following
treatment with control bsAbs binding only Trop-2 (TF12) or CD3
[(20)-3s], or with (E1)-3s in the absence of BxPC3 cells. The
uptake of Trop-2 by T cells coincided with its reduction on BcPC3
cells (FIG. 26). During the short incubation time, the T cells
(97.5% live) and BxPC3 (94.5% live) remained at high viability,
indicating that the T cells acquired the tumor antigens by
trogocytosis and not by adhering to membrane fragments of dead
cells (not shown). Trogocytosis mediated by (E1)-3s was
bidirectional, because T-cell membrane components were transferred
to BxPC3 cells, as demonstrated for CD4 (data not shown).
Example 23
Bispecific Anti-CD3.times.Anti-Trop-2 Antibodies and Cytokine
Release
As discussed in the Examples above, we studied the effects of
interferon-.alpha.(IFN.alpha.) on (E1)-3s-mediated T-cell killing
of human gastric and pancreatic cancer cell lines. T-cell
activation, cytokine induction, and cytotoxicity were evaluated ex
vivo using peripheral blood mononuclear cells (PBMC) or T cells
with NCI-N87 gastric cancer as target cells. In the presence of
target cells and PBMCs, (E1)-3s did not cause excess cytokine
production. When combined with (E1)-3s, peginterferonalfa-2a--which
alone did not increase T-cell activation or raise cytokine levels
over baseline--increased CD69 expression but did not significantly
increase cytokine induction. IFN.alpha. enhanced the therapeutic
efficacy of (E1)-3s without increasing the production of other
cytokines to levels that could induce cytokine release syndrome
(CRS). Adjuvant therapy with IFN.alpha., or other cytokines, might
be universally applicable for enhanced efficacy of T-cell
immunotherapy.
Methods
Cytokine release was measured ex vivo using 5.times.10.sup.5
cells/0.5 mL/well of either NCI-N87, which were allowed to attach
overnight, or Raji. Freshly isolated PBMCs (5.times.10.sup.6
cells/0.4 mL/well) were added to each well. Treatments (100 .mu.L,
10.times.) comprising (19)-3s, 19-3 BiTE, (E1)-3s,
peginterferonalfa-2a, or (E1)-3s plus peginterferonalfa-2a were
added to 0.1 nmol/L for each reagent. Alternatively, titrations
ranging from 1 pmol/L to 10 nmol/L were used for dose-response
studies. Following a 20-hour incubation at 37.degree. C. with
gentle shaking, the supernatant fluid was diluted 1:2 (or greater
when necessary) and the concentrations of TNF.alpha., IFNg, IL2,
IL6, and IL10 measured using Single-Analyte ELISArray kits
(Qiagen), following the manufacturer's protocol.
Results
A Trop-2.times.CD3 BiTE (or equivalent) was not available for
comparison with (E1)-3s. However, the availability of both (19)-3s,
which has the same (X)-3s molecular configuration as (E1)-3s, and
19-3 BiTE, which has the identical amino acid sequence as the
CD19.times.CD3 BiTE, blinatumomab, enabled a head-to-head
comparison to evaluate the relative cytokine-inducing potency of
the two bsAb formats.
Initially, titrations of (19)-3s and 19-3 BiTE were added to
mixtures of PBMCs (two independent donors), and Raji NHL cells and
the levels of TNF.alpha., IFN.gamma., and IL6 were measured after
20 hours (not shown). Minimal cytokine levels were detected from
PBMCs alone, even with the addition of a bsAb. However, because of
a mixed lymphocyte reaction occurring between Raji and the donor
PBMCs (stronger for donor A), cytokine levels in untreated cell
mixtures were elevated for each of TNF.alpha. (200 and 50 pg/mL),
IFN.gamma. (600 and 200 pg/mL), and IL6 (190 and 220 pg/mL). The
levels of TNF.alpha. and IL6 were increased above those of
untreated only at .gtoreq.1 nmol/L (19)-3s. Apparently, (19)-3s
inhibited TNF.alpha. and IL6 production at lower concentrations. In
comparison, TNF.alpha. and IL6 were elevated to >1,000 pg/mL at
all concentrations of 19-3 BiTE tested (.gtoreq.1 pmol/L). The
levels of IFN.gamma. were not increased significantly by (19)-3s,
whereas 19-3 BiTE showed a dose-dependent increase to >2,000
pg/mL.
For all further comparisons, agents were tested at 0.1 nmol/L,
which is approximately what has been used in similar studies with
BiTE (Brandl et al., Cancer Immunol Immunother 2007, 56:1551-63).
We compared the levels of TNF.alpha., IFN.gamma., IL2, IL6, and
IL10 induced by 0.1 nmol/L (19)-3s or 19-3 BiTE from Raji mixed
with PBMCs, using 4 different donors (FIG. 27A). With each of the 4
donors, the levels of each of the five cytokines were significantly
higher with 19-3 BiTE, compared with (19)-3s. The mean TNF.alpha.
concentration with 19-3 BiTE (2,284.+-.1,483 pg/mL) was 8-fold
higher (P=0.0001) than that with (19)-3s (280.+-.188 pg/mL).
Treatment with 19-3 BiTE, compared with (19)-3s, resulted in levels
of IFN.gamma. (3,002.+-.560 pg/mL vs. 416.+-.169 pg/mL), IL2
(13,635.+-.2,601 pg/mL vs. 1,024.+-.598 pg/mL), IL6 (981.+-.364
pg/mL vs. 168.+-.96 pg/mL), and IL10 (4,006.+-.2,520 pg/mL vs.
493.+-.242 pg/mL) that were 7-, 13-, 6-, and 8-fold higher for 19-3
BiTE, respectively (P<0.0001 for each). These results indicate
that the (X)-3s bsAb format is a considerably less potent inducer
of cytokine release, compared with the BiTE format.
In general, (E1)-3s in the presence of PBMCs and target cells
caused even less cytokine production than (19)-3s, because there is
no mixed lymphocyte reaction to elevate the baseline levels (FIG.
27B). Levels remained low for the proinflammatory cytokines
IFN.gamma. (<100 pg/mL), TNF.alpha. (<100 pg/mL), and IL2
(<250 pg/mL) with 4 of 5 donors. IL6 was low (<400 pg/mL) in
3 of 5 donors, and moderate (800-1,100 pg/mL) in donors D-2 and
D-5. Donor D-2 also responded to the (E1)-3s more than the others
for IFN.gamma. (1,000 pg/mL) and TNF.alpha. (190 pg/mL). IL10, an
anti-inflammatory cytokine, was significantly (P<0.0001)
elevated by (E1)-3s to >1,200 pg/mL in 3 of 5 donors. Of note,
donor D-2, who had a uniquely potent proinflammatory response,
produced relatively low levels of IL10 (230 pg/mL) after treatment
with (E1)-3s. Peginterferonalfa-2a alone did not increase the level
of any cytokine over background. Addition of peginterferonalfa-2a
to (E1)-3s consistently increased IFN.gamma. (.about.1.5-3-fold)
over (E1)-3s alone. For the rest of the cytokines, there was an
apparent trend for a
moderately increased production with the combination; however, a
consistent effect was not observed.
Example 24
In Vitro Cytotoxicity Induced by Bispecific
Anti-CD3.times.Anti-Trop-2 Antibodies
Further studies were performed to examine in vitro cytotoxicity
induced by anti-CD3.times.anti-Trop-2 bispecific antibodies.
Methods
Freshly-isolated CD8.sup.+ T cells were incubated for 24 h with 0.1
nM peginterferonalfa-2a, 0.1 nM 20*-2b, or media only. Treated or
untreated T cells and PKH67 green fluorescent-labeled NCI-N87 cells
were combined at a 5:1 ratio (5.times.10.sup.4 target cells and
2.5.times.10.sup.5 effector cells/well) in 48-well plates
containing serial dilutions of (E1)-3s in triplicate.
Peginterferonalfa-2a or 20*-2b were maintained at 0.1 nM in the
appropriate cell mixtures. Plates were incubated for 48 h at
37.degree. C. Suspension cells were removed and the attached cells
were detached with trypsin-EDTA and combined with the corresponding
suspension. Cells were washed and resuspended in 1% BSA-PBS
containing 30,000 COUNTBRIGHT.TM. Absolute Counting Beads (Life
Technologies) and 1 .mu.g/mL of 7-AAD. Total live target cells
(7-AAD.sup.-/PKH67.sup.+) were counted by flow cytometry. For each
sample, 8,000 COUNTBRIGHT.TM. beads were counted as a normalized
reference. The specific lysis (%) was calculated using the formula:
[1-(A.sub.1/A.sub.2)].times.100, where A.sub.1 and A.sub.2
represent the number of viable target cells in the test and
untreated samples, respectively. Statistical significance
(P.ltoreq.0.05) was determined for IC.sub.50 (the concentration
resulting in 50% lysis), EC.sub.50 (50% effective concentration)
and lysis.sup.max (maximal target cell lysis) by F-test on
non-linear regression (sigmoidal dose-response) curves with Prism
software.
Results
To evaluate redirected T-cell killing of Trop-2-expressing tumor
cells, CD8.sup.+ T cells were mixed with NCI-N87 cells in the
presence or absence of IFN-.alpha.2 (0.1 nM peginterferonalfa-2a or
20*-2b) along with titrations of (E1)-3s (FIG. 28). Considerable
variability in T-cell potency was observed among donors (FIG. 28A,
FIG. 28B). With a donor of very active T cells, (E1)-3s mediated a
highly potent (IC.sub.50=0.37 pM; lysis.sup.max=77.1%) T-cell lysis
of NCI-N87 cells, and inclusion of peginterferonalfa-2a enhanced
its activity, improving the IC.sub.50 (0.14 pM; P=0.0001) by more
than 2.5 fold and increasing lysis.sup.max (84.0%; P<0.0001)
(FIG. 28A). NCI-N87 was only weakly sensitive to the direct actions
of IFN-.alpha. (peginterferonalfa-2a IC.sub.50=>10 nM, data not
shown), and inhibited <10% by 0.1 nM peginterferonalfa-2a in the
absence of (E1)-3s. The more potent form of IFN.alpha., 20*-2b,
consisting of 4 IFN-.alpha. molecules fused to a bivalent anti-CD20
mAb by DNL.RTM., enhanced the potency of (E1)-3s by more than
7-fold (IC.sub.50=0.05 pM; P<0.0001). At 0.1 nM, 20*-2b
inhibited NCI-N87 by 12.6% in the absence of (E1)-3s. The 20*-2b
was included only to show enhanced activity with another (more
potent) form IFN-.alpha., and that the effect is not restricted to
peginterferonalfa-2a. The anti-CD20 mAb moiety is not functional in
this experiment. In a similar assay using very weak donor T cells,
(E1)-3s was considerably less potent (EC.sub.50=39 pM;
lysis.sup.max=21%); however, addition of peginterferonalfa-2a
enhanced the potency by >25 fold (EC.sub.50=1.4 pM; P=0.0008)
(FIG. 28B). Potent (E1)-3s-mediated T-cell killing also was
observed for the human pancreatic cancer line, BxPC3 (IC.sub.50=0.4
pM); however, the effect of adding IFN-.alpha. was not evaluated
with this cell line (not shown).
Example 25
Dose-Response Curves for T Cell Activation by
Anti-CD3.times.Anti-Trop-2 Bispecific Antibodies
Addition of 0.1 nM peginterferonalfa-2a increased CD69 upregulation
on T cells treated with (E1)-3s moderately, but significantly. For
(E1)-3s dose-response experiments measuring % CD69.sup.+ T cells,
the EC.sub.50 was lowered from 26 pM to 16 pM (P<0.0001) for
CD4.sup.+ T cells, and from 11 pM to 6 pM (P=0.0204) for CD8.sup.+
T cells in the presence of IFN-.alpha. (FIG. 29A).
Peginterferonalfa-2a combined with (E1)-3s resulted in more
CD69.sup.+ cells (FIG. 29B, FIG. 29C, P<0.0001), and also, the
activated cells had significantly higher CD69 expression with
IFN-.alpha. (FIG. 29B, FIG. 29D; MFI=907 vs 726; P<0.0001).
Peginterferonalfa-2a induced minimal CD69 expression in the absence
of (E1)-3s. Likewise, (E1)-3, either alone or in combination with
peginterferonalfa-2a, did not activate T cells in the absence of
target cells.
Example 26
Extended In Vivo Survival with (E1)-3s is Augmented with
IFN-.alpha.
The preliminary data on in vivo survival reported in Example 3
above were further extended to as long as 126 days. As shown below,
the combination of (E1)-3s with IFN-.alpha. provided the greatest
benefit for animals bearing Trop-2.sup.+ xenograft tumors.
Methods
Female 4-8-week old NOD/SCID mice (Charles River, Wilmington,
Mass.) were injected s.c. with a mixture of 5.times.10.sup.6 tumor
cells (Capan-1 or NCI-N87) and T cells (2.5.times.10.sup.6)
combined with an equal volume of matrigel. Therapy began 1 h later
by i.v. injection, as per the BiTE methodology (Dreier et al.,
2003, J Immunol 170:4397-402). Treatment regimens, dosages, and
number of animals in each experiment are described in the figure
legends. Tumor volume was determined twice weekly by measurements
in two dimensions using calipers, with volumes defined as:
L.times.w.sup.2/2, where L is the longest dimension of the tumor
and w the shortest.
Statistical analysis of tumor growth was based on area under the
curve (AUC). Profiles of individual tumor growth were obtained
through linear-curve modeling. An F-test was employed to determine
equality of variance between groups prior to statistical analysis
of growth curves. A Critical Z test on the survival data identified
any outliers within a given treatment group with P.ltoreq.0.05
censored from the final data analysis. A two-tailed t-test was used
to assess statistical significance between the various treatment
groups and controls, except for the untreated control, where a
one-tailed t-test was used. Additionally, efficacy was determined
by log-rank using Prism software on Kaplan-Meier curves using
survival surrogate endpoints as time for tumor progression to 1.0
cm.sup.3. Significance was considered at P.ltoreq.0.05 for all
comparisons.
Results
In vivo efficacy with human pancreatic cancer was evaluated with
Capan-1 xenografts. In the first study, treatment with a
combination of (E1)-3s and peginterferonalfa-2a [median survival
time (MST) >59 days] was superior to all other treatments
(P<0.0007, log-rank), including (E1)-3s (MST=50 days) or
peginterferonalfa-2a (MST=53 days) alone (FIG. 30A). Even with the
omission of T cells, peginterferonalfa-2a extended survival (MST=45
days, P=0.0059 vs saline, log-rank), indicating direct action on
the tumor cells. However, peginterferonalfa-2a was more effective
in the presence of T cells (P=0.0260, AUC), suggesting stimulation
of T cells by IFN-.alpha.. TF12, which binds target but not T
cells, did not affect tumor growth or survival. A repeat
experiment, using T cells from a different donor, confirmed the
results of the first study (FIG. 30B). The second study continued
until all groups reached their MST. As in the initial experiment,
the combination of (E1)-3s and peginterferonalfa-2a (MST=119.5
days) was superior to all other groups in terms of both tumor
growth inhibition and overall survival (P=0.0475 vs (E1)-3s alone;
P<0.0001 vs all other groups; log-rank). (E1)-3s (MST=68 days)
was superior (P=0.0373, AUC over 29 days) to peginterferonalfa-2a
with T cells (MST=53 days) and to T cells alone (MST=37.5 days;
P=0.0014 log-rank).
For the NCI-N87 gastric cancer xenograft model (FIG. 30C), the
combination of (E1)-3s and peginterferonalfa-2a (MST>88 days)
was superior to (E1)-3s alone (MST=49 days; P=0.0007, log-rank).
Compared to the control group with only T cells (MST=32 days),
peginterferonalfa-2a alone with T cells provided only a minor, but
significant, survival advantage (MST=35 days; P=0.0276). (E1)-3s
plus peginterferonalfa-2a without T cells did not improve survival
significantly.
The antigen density measured for NCI-N87 [247,000(.+-.65,000)
Trop-2/cell] and Capan-1 [157,000 (.+-.37,000) Trop-2/cell] was not
significantly different. Compared to NCI-N87, Capan-1 cells were
>5-fold more sensitive (IC.sub.50=2 nM vs.>10 nM) to direct
inhibition by peginterferonalfa-2a in vitro (not shown). (E1)-3s
does not cross-react with mouse Trop-2 or CD3 (not shown), and
NOD-SCID mice are T-cell deficient.
Discussion
This section discusses results presented in Examples 23-26. We
described in Examples 1 and 2 above the use of the (X)-3s bsAb
format for redirecting T cell-mediated therapy of both
hematopoietic and solid tumors using several example constructs,
including (E1)-3s, (19)-3s and (20)-3s. In one in vivo experiment
from that study, where Capan-1 xenografts were treated with
(E1)-3s, we included groups with peginterferonalfa-2a, because
prior (unpublished) data showed that Capan-1 was inhibited by
IFN-.alpha.. The striking enhancement observed with the addition of
IFN-.alpha. spurred further investigation, leading to the current
studies. The results of studies with T cell redirecting bispecific
antibodies, in combination with peginterferonalfa-2a are reported
herein. The studies were extended until all groups reached their
MST, confirming that IFN-.alpha. can enhance the in-vivo efficacy
of T-cell killing of an IFN-.alpha.-sensitive cell line.
IFN-.alpha. also can enhance T-cell-mediated killing of a cell line
that is weakly sensitive to the direct actions of IFN-.alpha..
These in vivo studies were performed following methods, including
dosing and schedules, typically used with BiTE constructs.
Flieger and colleagues demonstrated that in-vitro killing by
CD3.sup.+CD56.sup.+ NK-T cells, which were expanded ex vivo and
redirected with an EpCAM.times.CD3 BiTE (MT110), was enhanced with
either IFN-.alpha. or IL-2 (Flieger et al., 2000, Cancer Immunol
Immunother 49:441-8). However, even in the absence of the bsAb,
IFN-.alpha. significantly inhibited the target cells. Since a
control to evaluate potential direct effects of IFN-.alpha. on
target cells was lacking, the extent to which the enhanced
cytotoxicity was due to IFN-.alpha. stimulating NK-T cells,
compared to direct inhibition of target cells, could not be
determined. Therefore, we measured the sensitivity to IFN-.alpha.
for both target cells and included groups with peginterferonalfa-2a
only, both in the presence and absence of pan-T cells. For Capan-1
tumors, which were more sensitive to IFN-.alpha. in vitro,
peginterferonalfa-2a improved survival in the absence of T cells,
and even more so in the presence of T cells, indicating that
IFN-.alpha. acted on both Capan-1 as well as T cells in this model.
In the absence of T cells, peginterferonalfa-2a did not improve
survival of mice bearing NCI-N87 xenografts, which were weakly
sensitive to IFN-.alpha. in vitro, indicating that the enhancement
with IFN-.alpha. was due primarily to its actions on T cells. The
mechanism of the observed T-cell enhancement by IFN-.alpha. is
unclear. The increase in CD69 expression attributed to IFN-.alpha.
was moderate, but significant, suggesting that the cytokine may
potentiate T-cell activation induced with the bsAb. Additionally,
IFN-.alpha. specifically increased (up to 3-fold) the release of
IFN-.gamma., which is considered the chief cytotoxic cytokine
produced by cytotoxic T cells, whereas none of the other cytokines
measured increased consistently.
Combination therapy with IFN-.alpha. and a T-cell-redirecting bsAb
has not been investigated clinically, or even in animal models.
However, IL-2 was combined with a F(ab').sub.2 fragment of an
anti-CD3/EpCAM quadroma in a clinical trial (Kroesen et al., 1997,
Cancer Immunol Immunother 45:203-6), but treatment was limited due
to considerable toxicity most likely caused by induction of
secondary cytokines, known as CRS or cytokine storm. Systemic
administration of IL-2 is known to induce a cytokine storm (Panelli
et al., 2004, J Transl Med 2:17), and the severity of adverse
events associated with CRS, such as with the TGN1412 catastrophic
trial, are correlated with IL-2 release (Eastwood et al., 2013, Br
J Clin Pharmacol 76:299-315). Although it is not without side
effects, immunotherapy with IFN-.alpha., which is not produced by T
cells, is not typically associated with cytokine storm.
CRS is a risk associated with immunotherapy using any T-cell
directed mAb (e.g., Okt3) or bsAb, including BiTE (Klinger et al.,
2012, Blood 119:6226-33). However, not all bsAb formats necessarily
have the same risk. Brandl et al. reported cytokine induction with
blinatumomab, where response levels of IL-2, IL-6, IFN-.gamma., and
TNF-.alpha. were variable among donors and typically peaked at
>1 ng/mL, with some donors reaching levels as high as 5 ng/mL
(Brandl et al., 2007, Cancer Immunol Immunother 56:1551-63). We
lacked a suitable BiTE, or equivalent construct, for direct
comparison with (E1)-3s. However, we were able to compare the
relative cytokine-inducing potency between the (X)-3s and BiTE
formats, using a CD19.times.CD3 BiTE (identical sequence as
blinatumomab) and (19)-3s made by DNL.RTM.. The 19-3 BiTE induced
similar cytokine levels as reported by Brandl and colleagues under
similar conditions. The levels of the five cytokines measured were
7-13-fold higher for 19-3 BiTE, compared to those of (19)-3s. The
use of foreign lymphoma cells (Raji) caused a mixed lymphocyte
reaction, which increased the baseline cytokine levels,
particularly for IL-2. BiTE, but not (19)-3s, increased the
cytokine levels well above the mixed lymphocyte baseline level.
Using NCI-N87 gastric carcinoma cells as the target for (E1)-3s did
not increase baseline cytokine levels. We observed an expected
variability in donor response to (E1)-3s; however, the resulting
cytokine levels were even lower than those induced by (19)-3s,
particularly for TNF-.alpha. and IFN-.gamma., which were <100
pg/mL. Nevertheless, one of five donors had elevated levels
(.about.1 ng/mL) of IFN-.gamma. and IL-6. Addition of IFN-.alpha.
(peginterferonalfa-2a) to (E1)-3s increased IFN-.gamma. 2-3-fold,
but did not consistently affect the levels of the other cytokines.
These results suggest that compared to other constructs, such as
BiTE, the (X)-3s bsAb format is less likely to induce CRS, and the
addition of IFN-.alpha. to a therapeutic regimen does not increase
this risk.
We observed considerable variability in the potency of donor T
cells. The in vitro results shown in FIG. 28 represent the most and
least active T cells that we have tested, with a 100-fold
difference in potency (IC.sub.50=0.37 pM vs. 39 pM) for killing
NCI-N87; however, an IC.sub.50=1-5 pM is most representative
(>10 donors) and the low-activity T cells was atypical. Notably,
lysis with the weaker T cells was augmented by IFN-.alpha. more
than with the potent T cells.
EpCAM is a widely exploited TAA that is overexpressed in many
carcinomas. However, the heterogeneous expression of EpCAM in
carcinomas and the fact that EpCAM is not tumor-specific, since it
is expressed on most normal epithelia, raise concerns that
immunotherapy directed towards EpCAM could have severe side effects
(Balzar et al., 1999, J Mol Med (Berl) 77:699-712; Momburg et al.,
1987, Cancer Res 47:2883-91). Like EpCAM, Trop-2 is highly
expressed in diverse carcinomas, but its expression in normal
tissues is under debate. Several reports indicate that, in contrast
to tumor cells, somatic adult tissues show little or no Trop-2
expression, which is invariably upregulated in tumors, regardless
of baseline expression in normal tissues (Wang et al., 2008, Mol
Cancer Ther 7:280-5; Zhang et al., 1997, Science 276:1268-72).
However, recent evidence indicates expression of Trop-2 on
epithelia of several normal tissues (Trerotola et al., 2013,
Oncogene 32:222-33). Nonetheless, expression of Trop-2 in
Cynomolgus monkeys did not result in toxicities after
administrations of reasonably high doses of hRS7 (humanized
anti-Trop-2) conjugated with SN-38 as an antibody-drug conjugate
(ADC) (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Further,
in clinical studies with this anti-Trop-2 ADC, no increased normal
organ toxicity other than manageable neutropenia and diarrhea,
expected from the drug (a metabolite of irinotecan), was observed
at therapeutic doses (Starodub et al., Proceedings of the 105th
Annual Meeting of the American Association for Cancer Research.
2014 (abstr CT206)). Thus, immunotherapy, including
T-cell-redirected therapy, using Trop-2 for tumor targeting, is
expected to have a similar, or greater, therapeutic index compared
to similar regimens targeting EpCAM.
This is the first report of trogocytosis between target tumor and T
cells mediated by a bsAb. This finding demonstrates that the
target/T-cell conjugates induced with (E1)-3s have functional
immunologic synapses. We observed a similar bi-directional
trogocytosis between B cells and T cells, which was mediated by
(19)-3s (unpublished data), and believe this is likely a common
phenomenon with T-cell redirecting bsAbs.
Example 27
Further Studies with E1-3 Bispecific Antibodies Summary
A T-cell redirecting bispecific tandem scFv, E1-3, was produced as
described in Example 19 above, using the variable domains of hRS7
(humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). The studies
reported in this Example continue and expand on the results shown
in Examples 20-25. Any discrepancies between the instant reported
results and those shown in Examples 20-25 are based on the
collection of additional data. T-cell activation, proliferation,
cytokine induction and cytotoxicity were evaluated ex vivo using
PBMCs or purified T cells with human pancreatic (Capan-1 and
BxPC-3) and gastric (NCI-N87) cancer cell lines as target cells. In
vivo activity was assayed with NCI-N87 xenografts that were
inoculated s.c. in a mixture with twice the number of human PBMCs
and matrigel.
Results
In the presence of target cells and PBMCs, E1-3 potently induced
T-cell activation, proliferation and a dose-dependent cytokine
production of IL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7
ng/mL), TNF-.alpha. (>1 ng/mL) and IFN-.gamma. (>50 ng/mL).
Using 3-5 different T cell donors, E1-3 mediated a highly potent
T-cell lysis of BxPC-3 [IC.sub.50=0.09(.+-.0.04) pM], Capan-1
[IC.sub.50=1.2(.+-.1.1) pM] and NCI-N87 [IC.sub.50=1.2(.+-.1.2) pM]
target cells in vitro. In vivo, two 50-.mu.g doses of E1-3 given
three days apart cured 6 of 8 mice bearing NCI-N87 xenografts
(P<0.0001; Log-Rank). Tumors in the control group (PBMCs only)
reached the endpoint (TV>1 cm.sup.3) with a median of 39.5 days.
Seven of 8 animals had not reached the endpoint, with six of the
mice remaining tumor-free in the E1-3 group when the experiment was
terminated after 176 days.
T-Cell Activation and Proliferation--
Purified CD8.sup.+ T cells were mixed 5:1 with NCI-N87 cells,
treated for 18 h with 0.01 nM E1-3 and analyzed by flow cytometry.
CD69 was upregulated by E1-3 in the presence of target cells (not
shown). Treatments with omission of E1-3 or NCI-N87 target cells
did not induce CD69 expression (not shown). Additionally, T cells
experienced an increase in forward (FSC) and side scattering (SSC)
after culture in the presence of E1-3 and target cells (not shown).
T-cell proliferation was evident after three days (P<0.005, data
not shown).
Cytokine Release--
The ability of E1-3 bispecific tandem scFv to induce release of
cytokines IFN-.gamma., TNF-.alpha., IL-2, IL-6 and IL-10 as a
function of dosage was determined. As shown in FIG. 31, the E1-3
bispecific antibody effectively induced cytokine release in the
picomolar concentration range.
In Vitro T-Cell Mediated Killing--
The ability of E1-3 to induce T-cell mediated killing of target
pancreatic and gastric cancer cells was determined in the presence
of purified CD8.sup.+ T cells (1.2.times.10.sup.5/well). An
exemplary dose-response curve using T-cells from a representative
donor are shown in FIG. 32. In this experiment, the IC.sub.50
values for E1-3 were 0.6 pM for Capan-1, 0.1 pM for BxPC-3 and 0.3
pM for NCI-N87.
In Vivo Anti-Tumor Effects of E1-3--
Nude mice bearing NCI-N87 xenografts were treated with two 50-.mu.g
doses of E1-3 given three days apart. The treatment (FIG. 33A)
cured 6 of 8 mice bearing the human gastric cancer xenografts
(P<0.0001; Log-Rank). In comparison with tumors in the control
group (treated with PBMCs only) reached the endpoint (TV>1
cm.sup.3) with a median of 39.5 days (FIG. 33B). When the study was
terminated after 176 days, seven of eight animals in the E1-3 group
had not reached the endpoint.
Conclusions
The studies above show that Trop-2 is an attractive target for
T-cell-mediated killing of pancreatic, gastric and other epithelial
cancers. The E1-3 anti-Trop-2.times.anti-CD3 bispecific antibody
induced potent T-cell activation and cytokine production. E1-3 was
highly effective at killing solid tumors in vitro and in vivo.
All of the COMPOSITIONS and METHODS disclosed and claimed herein
can be made and used without undue experimentation in light of the
present disclosure. While the compositions and methods have been
described in terms of preferred embodiments, it is apparent to
those of skill in the art that variations maybe applied to the
COMPOSITIONS and METHODS and in the steps or in the sequence of
steps of the METHODS described herein without departing from the
concept, spirit and scope of the invention. More specifically,
certain agents that are both chemically and physiologically related
may be substituted for the agents described herein while the same
or similar results would be achieved. All such similar substitutes
and modifications apparent to those skilled in the art are deemed
to be within the spirit, scope and concept of the invention as
defined by the appended claims.
SEQUENCE LISTINGS
1
120144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 245PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 2Cys Gly His Ile Gln Ile
Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly 1 5 10 15 Tyr Thr Val Glu
Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe 20 25 30 Ala Val
Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 45
317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 421PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 4Cys Gly Gln Ile Glu Tyr
Leu Ala Lys Gln Ile Val Asp Asn Ala Ile 1 5 10 15 Gln Gln Ala Gly
Cys 20 550PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys
His Asn Ile Gln Ala 1 5 10 15 Leu Leu Lys Asp Ser Ile Val Gln Leu
Cys Thr Ala Arg Pro Glu Arg 20 25 30 Pro Met Ala Phe Leu Arg Glu
Tyr Phe Glu Arg Leu Glu Lys Glu Glu 35 40 45 Ala Lys 50
655PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Met Ser Cys Gly Gly Ser Leu Arg Glu Cys Glu
Leu Tyr Val Gln Lys 1 5 10 15 His Asn Ile Gln Ala Leu Leu Lys Asp
Ser Ile Val Gln Leu Cys Thr 20 25 30 Ala Arg Pro Glu Arg Pro Met
Ala Phe Leu Arg Glu Tyr Phe Glu Arg 35 40 45 Leu Glu Lys Glu Glu
Ala Lys 50 55 723PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Cys Gly Phe Glu Glu Leu Ala Trp Lys Ile
Ala Lys Met Ile Trp Ser 1 5 10 15 Asp Val Phe Gln Gln Gly Cys 20
851PRTHomo sapiens 8Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys His
Asn Ile Gln Ala 1 5 10 15 Leu Leu Lys Asp Val Ser Ile Val Gln Leu
Cys Thr Ala Arg Pro Glu 20 25 30 Arg Pro Met Ala Phe Leu Arg Glu
Tyr Phe Glu Lys Leu Glu Lys Glu 35 40 45 Glu Ala Lys 50 954PRTHomo
sapiens 9Ser Leu Lys Gly Cys Glu Leu Tyr Val Gln Leu His Gly Ile
Gln Gln 1 5 10 15 Val Leu Lys Asp Cys Ile Val His Leu Cys Ile Ser
Lys Pro Glu Arg 20 25 30 Pro Met Lys Phe Leu Arg Glu His Phe Glu
Lys Leu Glu Lys Glu Glu 35 40 45 Asn Arg Gln Ile Leu Ala 50
1044PRTHomo sapiens 10Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Gly Gln Gln Pro Pro
Asp Leu Val Asp Phe Ala Val 20 25 30 Glu Tyr Phe Thr Arg Leu Arg
Glu Ala Arg Arg Gln 35 40 1144PRTHomo sapiens 11Ser Ile Glu Ile Pro
Ala Gly Leu Thr Glu Leu Leu Gln Gly Phe Thr 1 5 10 15 Val Glu Val
Leu Arg His Gln Pro Ala Asp Leu Leu Glu Phe Ala Leu 20 25 30 Gln
His Phe Thr Arg Leu Gln Gln Glu Asn Glu Arg 35 40 1244PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Thr His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1
5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
1344PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Ser Lys Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 1444PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 14Ser Arg Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val
Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 1544PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Ser His Ile Asn Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1
5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
1644PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Ser His Ile Gln Ile Pro Pro Ala Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 1744PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 17Ser His Ile Gln Ile Pro
Pro Gly Leu Ser Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val
Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 1844PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Ser His Ile Gln Ile Pro Pro Gly Leu Thr Asp Leu Leu Gln Gly Tyr 1
5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
1944PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Asn Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 2044PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 20Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Ala Tyr 1 5 10 15 Thr Val Glu Val
Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 2144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1
5 10 15 Ser Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
2244PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Asp Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 2344PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val
Leu Lys Gln Gln Pro Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 2444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1
5 10 15 Thr Val Glu Val Leu Arg Asn Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
2544PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Asn Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 2644PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 26Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val
Leu Arg Gln Gln Pro Pro Glu Leu Val Glu Phe Ala 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 2744PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1
5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Asp Phe
Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
2844PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Leu 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 2944PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 29Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val
Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ile 20 25 30 Val Glu
Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 3044PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1
5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe
Val 20 25 30 Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40
3144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Asp Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 3217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Asn Ile Glu Tyr Leu Ala Lys
Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala 3317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Gln
Leu Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10
15 Ala 3417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Gln Val Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 3517PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 35Gln Ile Asp Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
3617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Gln Ile Glu Phe Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 3717PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 37Gln Ile Glu Thr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
3817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Gln Ile Glu Ser Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 3917PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 39Gln Ile Glu Tyr Ile Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
4017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 4117PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 41Gln Ile Glu Tyr Leu Ala
Arg Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala
4217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Gln Ile Glu Tyr Leu Ala Lys Asn Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 4317PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 43Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Glu Asn Ala Ile Gln Gln 1 5 10 15 Ala
4417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Gln
Ala Ile Gln Gln 1 5 10 15 Ala 4517PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 45Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Asn Gln 1 5 10 15 Ala
4617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Asn 1 5 10 15 Ala 4717PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 47Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Leu
4817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ile 4917PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 49Gln Ile Glu Tyr Leu Ala
Lys Gln Ile Val Asp Asn Ala Ile Gln Gln 1 5 10 15 Val
5017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Tyr
Ala Ile His Gln 1 5 10 15 Ala 5117PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 51Gln Ile Glu Tyr Lys Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
5217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Gln Ile Glu Tyr His Ala Lys Gln Ile Val Asp His
Ala Ile His Gln 1 5 10 15 Ala 5317PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 53Gln Ile Glu Tyr Val Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
5418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn
Ala Ile Gln Gln 1 5 10 15 Ala Ile 5518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Leu
Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile Gln Leu 1 5
10
15 Ser Ile 5618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 56Leu Ile Glu Glu Ala Ala Ser Arg Ile
Val Asp Ala Val Ile Glu Gln 1 5 10 15 Val Lys 5718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Ala
Leu Tyr Gln Phe Ala Asp Arg Phe Ser Glu Leu Val Ile Ser Glu 1 5 10
15 Ala Leu 5817PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 58Leu Glu Gln Val Ala Asn Gln Leu Ala
Asp Gln Ile Ile Lys Glu Ala 1 5 10 15 Thr 5917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Phe
Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val 1 5 10
15 Phe 6018PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn
Ala Val Leu Lys 1 5 10 15 Ala Val 6118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 61Thr
Ala Glu Glu Val Ser Ala Arg Ile Val Gln Val Val Thr Ala Glu 1 5 10
15 Ala Val 6218PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 62Gln Ile Lys Gln Ala Ala Phe Gln Leu
Ile Ser Gln Val Ile Leu Glu 1 5 10 15 Ala Thr 6316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 63Leu
Ala Trp Lys Ile Ala Lys Met Ile Val Ser Asp Val Met Gln Gln 1 5 10
15 6424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp
Ala Val Ile Glu 1 5 10 15 Gln Val Lys Ala Ala Gly Ala Tyr 20
6518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Leu Glu Gln Tyr Ala Asn Gln Leu Ala Asp Gln Ile
Ile Lys Glu Ala 1 5 10 15 Thr Glu 6620PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Phe
Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val 1 5 10
15 Phe Gln Gln Cys 20 6717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Gln Ile Glu Tyr Leu Ala Lys
Gln Ile Pro Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala 6825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala 1 5 10
15 Val Ile Glu Gln Val Lys Ala Ala Gly 20 25 6925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 69Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Pro Asp Ala 1 5 10
15 Pro Ile Glu Gln Val Lys Ala Ala Gly 20 25 7025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 7125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 71Pro
Glu Asp Ala Glu Leu Val Arg Thr Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 7225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Asp Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 7325PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 73Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 7425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Pro Leu Lys Ala Val Gln Gln Tyr 20 25 7525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Glu Lys Ala Val Gln Gln Tyr 20 25 7625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Glu
Glu Gly Leu Asp Arg Asn Glu Glu Ile Lys Arg Ala Ala Phe Gln 1 5 10
15 Ile Ile Ser Gln Val Ile Ser Glu Ala 20 25 7725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Leu
Val Asp Asp Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn 1 5 10
15 Ala Ile Gln Gln Ala Ile Ala Glu Gln 20 25 7825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Gln
Tyr Glu Thr Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn 1 5 10
15 Ala Ile Gln Leu Ser Ile Glu Gln Leu 20 25 7925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Leu
Glu Lys Gln Tyr Gln Glu Gln Leu Glu Glu Glu Val Ala Lys Val 1 5 10
15 Ile Val Ser Met Ser Ile Ala Phe Ala 20 25 8025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Asn
Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp Lys Ile Ala Lys Met 1 5 10
15 Ile Val Ser Asp Ile Met Gln Gln Ala 20 25 8125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Val
Asn Leu Asp Lys Lys Ala Val Leu Ala Glu Lys Ile Val Ala Glu 1 5 10
15 Ala Ile Glu Lys Ala Glu Arg Glu Leu 20 25 8225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Asn
Gly Ile Leu Glu Leu Glu Thr Lys Ser Ser Lys Leu Val Gln Asn 1 5 10
15 Ile Ile Gln Thr Ala Val Asp Gln Phe 20 25 8325PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Thr
Gln Asp Lys Asn Tyr Glu Asp Glu Leu Thr Gln Val Ala Leu Ala 1 5 10
15 Leu Val Glu Asp Val Ile Asn Tyr Ala 20 25 8425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Glu
Thr Ser Ala Lys Asp Asn Ile Asn Ile Glu Glu Ala Ala Arg Phe 1 5 10
15 Leu Val Glu Lys Ile Leu Val Asn His 20 25 85330PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
85Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Ala Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 86330PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 86Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
8744PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 87Xaa Xaa Ile Xaa Ile Pro Pro Xaa Leu Xaa Xaa
Leu Leu Xaa Xaa Tyr 1 5 10 15 Xaa Val Xaa Val Leu Xaa Xaa Xaa Pro
Pro Xaa Leu Val Xaa Phe Xaa 20 25 30 Val Xaa Tyr Phe Xaa Xaa Leu
Xaa Xaa Xaa Xaa Xaa 35 40 8817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 88Xaa Xaa Xaa Xaa Xaa Ala Xaa
Xaa Ile Val Xaa Xaa Ala Ile Xaa Xaa 1 5 10 15 Xaa 8944PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
89Xaa His Ile Xaa Ile Pro Pro Gly Leu Xaa Glu Leu Leu Gln Gly Tyr 1
5 10 15 Thr Xaa Glu Val Leu Arg Xaa Gln Pro Pro Asp Leu Val Glu Phe
Ala 20 25 30 Xaa Xaa Tyr Phe Xaa Xaa Leu Xaa Glu Xaa Arg Xaa 35 40
9015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Leu Asn 1 5 10 15 917PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 91Asp Ala Ser Asn Leu Val Ser
1 5 929PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 92Gln Gln Ser Thr Glu Asp Pro Trp Thr 1 5
935PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Ser Tyr Trp Met Asn 1 5 9417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Gln
Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10
15 Gly 9515PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 95Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala
Met Asp Tyr 1 5 10 15 96119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 96Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115
9715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15 98107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 98Asp Ile Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp
Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50
55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala
Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn
Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
100 105 995PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Gly Gly Gly Gly Ser 1 5 10011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100His
His His His His His Gly Gly Gly Ser Gly 1 5 10 10121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 101Cys
Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile 1 5 10
15 Gln Gln Ala Gly Cys 20 1024PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 102Pro Lys Ser Cys 1
10355PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 103Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser His Ile Gln Ile 1 5 10 15 Pro Pro Gly Leu
Thr Glu Leu Leu Gln Gly Tyr Thr Val Glu Val Leu 20 25 30 Arg Gln
Gln Pro Pro Asp Leu Val Glu Phe Ala Val Glu Tyr Phe Thr 35 40 45
Arg Leu Arg Glu Ala Arg Ala 50 55 10429PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 104Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Glu Tyr 1 5 10
15 Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln Ala 20 25
1056PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 105His His His His His His 1 5
1061579DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 106tctagacaca ggccgccatc atgggatgga
gctgtatcat cctcttcttg gtagcaacag 60ctacaggtgt ccactccgac attcagctga
cccagtctcc atcctccctg tctgcatctg 120taggagacag agtcagcatc
acctgcaagg ccagtcagga tgtgagtatt gctgtagcct 180ggtatcagca
gaaaccaggg aaagccccta agctcctgat ctactcggca tcctaccggt
240acactggagt ccctgatagg ttcagtggca gtggatctgg gacagatttc
actctcacca 300tcagcagtct gcaacctgaa gattttgcag tttattactg
tcagcaacat tatattactc 360cgctcacgtt cggtgctggg accaaggtgg
agatcaaagg tggaggaggg tccggtggag 420gagggtctgg tggaggaggg
agccaggtcc agctgcagca atctgggtct gagttgaaga 480agcctggggc
ctcagtgaag gtttcctgca aggcttctgg atacaccttc acaaactatg
540gaatgaactg ggtgaagcag gcccctggac aagggcttaa atggatgggc
tggataaaca 600cctacactgg agagccaaca tatactgatg acttcaaggg
acggtttgcc ttctccttgg 660acacctctgt cagcacggca tatctccaga
tcagcagcct aaaggctgac gacactgccg 720tgtatttctg tgcaagaggg
gggttcggta gtagctactg gtacttcgat gtctggggcc 780aagggtccct
ggtcaccgtc tcctcaggtg gcggagggtc cgatatcaag ctgcagcagt
840ctggagcaga gctcgctcga ccaggagcta gtgtgaagat gtcatgtaaa
acaagtggct 900atactttcac ccggtacact atgcactggg tcaagcagcg
cccaggacag ggtctggaat 960ggatcggcta cattaacccc agcaggggat
ataccaacta caatcagaag ttcaaggata 1020aagccaccct gactaccgac
aagtcctcta gtacagctta tatgcagctg tcaagcctca 1080cttccgagga
ctctgcagtg tattactgcg ccagatatta cgacgatcat tattgtctgg
1140attactgggg ccagggaaca actctcacag tgtcctctgt cgaaggtggc
agtggagggt 1200caggtggcag cggagggtcc ggtggagtgg acgatatcca
gctgacccag tctcctgcca 1260ttatgagcgc ttccccaggc gagaaggtga
caatgacttg ccgggccagt tcaagcgtca 1320gctatatgaa ttggtatcag
cagaagtctg gaaccagtcc taaacgatgg atctatgaca 1380catctaaagt
ggcaagcggg gtcccataca ggttctctgg gagtggttca ggcactagct
1440attccctgac catttcctct atggaggccg aagatgcagc cacctattac
tgtcagcagt 1500ggagttcaaa tccactcacc ttcggagcag gcactaaact
ggaactcaag caccaccacc 1560accaccacta aggcggccg
1579107497PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 107Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys
Ala Ser Gln Asp Val Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser
Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85
90 95 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly
Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
Leu Gln Gln 115 120 125 Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala Ser
Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr Gly Met Asn Trp Val Lys 145 150 155 160 Gln Ala Pro Gly Gln Gly
Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr 165 170 175 Thr Gly Glu Pro
Thr Tyr Thr Asp Asp Phe Lys Gly Arg Phe Ala Phe 180 185 190 Ser Leu
Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu 195 200 205
Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly Gly Phe Gly 210
215 220 Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser Leu Val
Thr 225 230 235 240 Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu
Gln Gln Ser Gly 245 250 255 Ala Glu Leu Ala Arg Pro Gly Ala Ser Val
Lys Met Ser Cys Lys Thr 260 265 270 Ser Gly Tyr Thr Phe Thr Arg Tyr
Thr Met His Trp Val Lys Gln Arg 275 280 285 Pro Gly Gln Gly Leu Glu
Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly 290 295 300 Tyr Thr Asn Tyr
Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr 305 310 315 320 Asp
Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser 325 330
335 Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr
340 345 350 Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
Ser Val 355 360 365 Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly Gly Val 370 375 380 Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro 385 390 395 400 Gly Glu Lys Val Thr Met Thr
Cys Arg Ala Ser Ser Ser Val Ser Tyr 405 410 415 Met Asn Trp Tyr Gln
Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile 420 425 430 Tyr Asp Thr
Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly 435 440 445 Ser
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala 450 455
460 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu
465 470 475 480 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys His His
His His His 485 490 495 His 108107PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 108Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr
Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile
Lys 100 105 10915PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 109Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 1 5 10 15 110121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
110Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly
Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro
Thr Tyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu
Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu
Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly
Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly
Ser Leu Val Thr Val Ser Ser 115 120 1115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 111Gly
Gly Gly Gly Ser 1 5 112119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 112Asp Ile Lys Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys
Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115
11318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly 1 5 10 15 Val Asp 114104PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
114Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser
Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys
Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro
Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly
Thr Lys Leu Glu 100 11511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 115Lys Ala Ser Gln Asp Val
Ser Ile Ala Val Ala 1 5 10 1167PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 116Ser Ala Ser Tyr Arg Tyr
Thr 1 5 1179PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 117Gln Gln His Tyr Ile Thr Pro Leu Thr 1
5 1185PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 118Asn Tyr Gly Met Asn 1 5 11917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 119Trp
Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys 1 5 10
15 Gly 12012PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 120Gly Gly Phe Gly Ser Ser Tyr Trp Tyr
Phe Asp Val 1 5 10
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